Method of showing progression of AIDS in an ARC patient by treating with Tyr-Gly compositions

ABSTRACT

Purified human leukocyte dialysates are described for treatment of AIDS, ARC, and other immunodeficient conditions. The dialysates are purified by HPLC processes, and are made available in a form that is free of endotoxin and pyrogen. Methods are described for slowing the progression from ARC to AIDS and for alleviating symptoms of AIDS and ARC, by administration of the dialysates. Methods for treating candidiasis and for increasing immune system response to recall antigen, by administration of the dialysates, are also described.

This is a continuation-in-part based on the disclosure contained in U.S.patent application Ser. No. 902,683, filed 2 Sept. 1986, andsubsequently abandoned, which priority date is claimed herein. Thatapplication was a continuation-in-part based on U.S. patent applicationSer. No. 643,724, which subsequenty issued as U.S. Pat. No. 4,616,079,was filed 24 Aug. 1984, and priority is claimed as to such date. This isa divisional application, resulting from a restriction requirementimposed in U.S. patent application Ser. No. 183,905 (filed Apt. 20,1988), now abandoned imposed by Office Action dated Jan. 11, 1989.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention concerns cell-mediated immunity and pathologicalconditions associated with a deficiency in cell-mediated immunity. Suchconditions include, in particular, the Acquired Immune DeficiencySyndrome (AIDS), caused by the Human Immunodeficiency Virus (HIV), andAIDS-Related Complex (ARC). The invention also concerns otherimmunodeficient conditions.

A typical manifestation of cell-mediated immunity is the delayed typehypersensitivity ("DH") skin reaction. A DH skin reaction is observedwhen an appropriate antigen is injected subcutaneously. Within 24 to 48hours, local inflammation (erythema) and a swelling and thickening(induration) are observed in a sensitive individual. The degree ofsensitivity may be measured by the size and severity of the reaction.The DH reaction also presents characteristic histologicalfindings--specifically, perivascular infiltration of leukocytes andmonocytes in the inflamed area. The cells seen at the site of a DHreaction are derived from the peripheral blood leukocyte population.

The mechanisms of cell-mediated immunity are as yet incompletelyunderstood. It is known that the cells which mediate the response arecapable of responding in a variety of ways to a challenge from anantigen. These responses include: proliferation of cells bearingspecific sensitivity to a given antigen; the induction andmultiplication of cells mediating a variety of immune functions,including antibody production; and reactions against foreign cells andtumors.

The present invention relates to the discovery of (1) endogenousamplifiers of the immune system, which are isolated from dialyzedextracts of leukocytes, and synthetic similar products; and (2) methodsof using, and compositions containing, the amplifiers. These amplifiersprofoundly affect the quality and quantity of cell-mediated immunityresponses; and are useful in the treatment of AIDS, ARC, and otherclinical conditions characterized by inadequate reaction to antigens.

2. Other Background

Earlier Gottlieb patents

In Gottlieb U.S. Pt. No. 4,468,379, it was disclosed that endogenousmaterials exist that amplify the speed and magnitude of cell-mediatedimmune system response. These amplifier materials are distinguished fromso-called transfer factors in that amplifiers do not transfer to asubject an immune response to a mitogen or antigen to which the subjecthas not previously been exposed and is not concurrently exposed, whiletransfer factors are said to do so. Moreover, amplifiers nonspecificallyincrease cell-mediated immune system responses to mitogens and antigensto which the subject has previously been or concurrently is exposed,while transfer factors are specific to particular antigens.

The material designated "amplifier 1" in the '379 patent is now known bythe inventor to be a mixture of various things. They include (1) what isreferred to subsequently in the present patent application asYG-material, (2) what is referred to subsequently in the present patentapplication as YGG-material, (3) another as-yet undefined orincompletely defined amplifier, (4) various amino acid products, and (5)other materials. The foregoing materials occurred in amplifier 1 invarying relative proportions, depending on the identity of the bloodsample from which the sample of amplifier 1 was derived. The reason forthat variation is that the content of a human blood sample varies fromdonor to donor and even for the same donor from time to time, dependingon the state of the immune system of the donor. The fact that thecontent of amplifier 1 varied from sample to sample adversely affectedthe repeatability of experiments directed toward establishing theimmunological activity of amplifier 1. That in turn adversely affectedability to establish product identity, standard dosages, assays, and thelike for amplifier 1.

It was suggested in Gottlieb U.S. Pat. No. 4,616,079 (from which theparent of this application was divided and of which said application wasa continuation-in-part) that amplifiers appear to act on T-helper cells(T4 cells) in a way that causes them to produce chemical mediators(lymphokines) whose effect is to increase the sped and/or magnitude ofcell-mediated immune system response to antigens and other means ofactivating a cell-mediated immune system response. Indicia of thisresponse include DH reaction to recall antigens, production of IL-2 andgamma interferon, and potentiation of cytotoxic cells.

It is known that various diseases and pathological conditions, such asAIDS and ARC, as well as chemotherapy, radiation, and aging, depress theimmune system response. A result is increased susceptibility toopportunistic infections, malignancies, and other pathologicalconditions that a normal immune system would have confronted. Frequently(and for some conditions, invariably), the result is death.Administration of amplifiers provides a means of increasingcell-mediated immune system responsiveness, where the cell-mediatedimmune system remains sufficiently intact for it to respond to suchadministration.

Earlier Gottlieb patents describe means of extracting amplifiermaterials from human leukocyte dialysates by reverse-phase HPLCprocesses. However, until recently the inventor did not havesufficiently detailed information about the molecular structure of theconstituents of such purified dialysate fractions to permit him toidentify the structures of their immunologically active components. Inlarge part this was because a way had not yet been discovered to purifythe dialysates sufficiently to permit necessary analysis. In anapplication filed during the pendency of the parent applications of theinstant application, and now issued as Gottlieb U.S. Pat. No. 4,699,898,as well as in other related patent applications of the inventor, theinventor disclosed his discovery of Tyr--Gly and Tyr--Gly--Gly peptidesas immunologically active components in the partially purified dialysatefractions previously described in earlier Gottlieb patents, such asGottlieb U.S. Pat. No. 4,616,079 (of which this application is acontinuation-in-part).

The earlier Gottlieb patents may also be consulted for other generalbackground information on amplifiers and their use. In this regard,mention should also be made of Gottlieb EPO pat. app. pub. no. 0173889(12.03.86), which is based on both Gottlieb U.S. Pat. No. 4,699,898 andthe parent application of the instant application.

Coy

Coy U.S. Pat. No. 4,127,534 described tripeptides of the formTyr--X--Gly, where X is a D-aminoacid. Coy asserts that these productshave analgesic and related utility, as indicated by rat tail flick orother tests; he indicates that, accordingly, they may be used assubstitutes for such medications as aspirin and sedatives. Coy claimspharmaceutical compositions that contain a "therapeutically effectiveamount" of Tyr--X--Gly, including Tyr--D--Ala--Gly. Coy asserts in thebody of his specification that a therapeutically effective amount of theproduct for purposes of the disclosed utility is from 0.001 mg per kg ofbodyweight to 100 mg per kg of bodyweight, administered daily.(Extrapolated to an 80 kg person, this amounts to a daily dose ofapproximately 0.1 mg to 10 g; 0.1 mg is equivalent to approximately 300nanomoles, and 10 g is equivalent to approximately 0.03 moles.) Itshould be noted that the relevant language of Coy's specification is inthe present tense, indicating use of prophetic examples. (No therapeuticExamples are provided in the Coy specification, and no statements aboututility or dosage are made in the past tense.)

Coy does not assert any immunological use of the products. Coy does notdescribe use of the D-aminoacid group as a means of preventing cleavageof the Tyr--Cly bond by endogenous enzymes. Coy does not describe anyutility for doses of less than the aforesaid minimum daily amounts(0,001 mg/kg, 0.1 mg, and 300 nM).

Plotnikoff

Plotnikoff U.S. Pat. No. 4,537,878 discloses and claims the use ofendogenous endorphins and enkephalins to stimulate the immune system.The dosage amounts actually used in vivo (Plotnikoff's Examples VIII toXI) were from 1 microgram (ug) per kg to 50 ug/kg, single i.v. dose.Elsewhere, however, Plotnikoff refers to a therapeutic dose of from 1ug/kg to 30 mg/kg, and to a preferable dosage rate of from 0.01 fg/kg to250 ug/kg. No explanation is given for the inconsistencies, and no datain the specification indicates a reason why these latter dosage rateswere mentioned or claimed. (they do not appear in Examples or similardata.)

The molecular species whose use Plotnikoff discloses are the endogenousenkephalin pentapeptides Tyr--Gly--Gly--Phe--Leu andTyr--Gly--Gly--Phe--Met, and longer endorphin polypeptide extensionsthereof (extended from the C-terminal end). Plotnikoff does not discloseuse of any nonendogenous peptides, and does not disclose anythingconcerning use of dipeptides, tripeptides, or tetrapeptides. Plotnikoffdoes not indicate that Tyr--Gly or Tyr--Gly--Gly have any immunologicalor other utility. Plotnikoff does not show that any products hedescribes have utility in treating AIDS or ARC.

Schwartz

Schwartz et al., Biological inactivation of enkephalins and the role ofenkephalin-dipeptidyl-carboxypeptidase ("enkephalinase") asneuropeptidase, 29 Enkephalin Metabolism 1715 (1981), extensivelyreviews work that has been done in the field of enzymatic breakdown ofenkephalins. Schwartz summarizes the content of the paper as follows:

In this review it will be shown that enkephalins are rapidly hydrolyzedin vivo and that several peptidase activities have been identified whichare able to cleave these molecules to give various biologically-inactivefragments. (Emphasis added.)

Schwartz and the work summarized in the review teach that variousendogenous enzymes cleave (hydrolyze) the Gly--Phe, Gly--Gly, andTyr--Gly bonds of endogenous mammalian polypeptides, such asLeuenkephalin (Tyr--Gly--Gly--Phe--Leu) and Met-enkephalin(Tyr--Gly--Gly--Phe--Met), into what Schwartz alleges are "biologicallyinactive fragments." Such fragments include what Schwartz refers to asTyr--Gly, which in the context of the paper apparently means a dipeptidecontaining Tyr and Gly amino acid residues, in that order; but Schwartzdoes not indicate what side chains or other groups, if any, are attachedto the amino acid residues or what specific molecular structure ispresent. It is thus left unstated in the paper precisely what thedetailed structure is of the product referred to as Tyr--Gly.

Schwartz and the work summarized in the review also disclose variousmeans of inhibiting such enzymatic cleavage, including N-methylation ofthe Tyr residue; esterification, amidification, and alcoholation of theC-terminal carboxyl; insertion of a D-amino-acid residue (such as D-Ala)into the chain near the C-terminal end; and mixture with bacitracin,puromycin, bestatin, amastatin, or thiorphan. (It is also known inpharmaceutical art, although not discussed in Schwartz, to bind orcomplex an enzyme-inhibiting agent to a therapeutically active molecule,so that the agent will preferentially bind to the active site on theenzyme that is to be inhibited, thereby preempting that site and thuskeeping the enzyme from hydrolyzing the molecule to be protected. Thisis exemplified by the use of the product sulbactam, a beta-lactamaseinhibitor used to protect ampicillin from beta-lactamase; thus, Unasyn™(Pfizer) is a mixture of sulbactam and ampicillin, while sultamicillinis ampicillin complexed or otherwise linked with sulbactam via an ester.It is also known, for example in the case of the synthetic penicillins,to introduce a large group (such as methyl) at a location on atherapeutically active molecule where there would otherwise be a spaceproviding a site for enzyme attachment, which results in hydrolysis. Theresult of occupying such a space is to inhibit enzymatic degradation ofthe molecule thus protected.)

The Schwartz paper does not mention any immunological activity or otherutility of the allegedly useless and biologically inactive fragmentsresulting from enzymatic action on enkephalins.

Delivery of drug via hydrolysis

It is known that a desired therapeutically active molecule may bedelivered by administering to a patient a different molecule thathydrolyzes, as a result of the action of endogenous enzymes, tofractions that include the desired therapeutically active molecule.Perhaps hetacillin is the best known example. Hetacillin breaks down inthe human body to ampicillin. A legal controversy ensuedinternationally, following the introduction of hetacillin, over whetherthe manufacture, use, and sale of hetacillin infringed patents onampicillin.

Commercial Tyr--Gly

Tyr--Gly is sold as a chemical reagent (L-tyrosylglycine) by SigmaChemical Co;, St. Louis, Mo., among others. Tyr--Gly is not sold inU.S.P. grade, and it is illegal under applicable laws to sell Tyr--Glyfor use as a pharmaceutical. Commercial grade Tyr--Gly is not consideredfree of pyrogens, endotoxin, and other pharmaceutically unacceptableconstituents. The presence of such pyrogens, endotoxin, and otherpharmaceutically unacceptable constituents makes a product unacceptablefor use as a drug, as that term is defined by federal statute, bothunder generally recognized medical principles and under FDA regulations.To the extent of the inventor's knowledge, no pharmaceuticalpreparations of this product are or have been available.

Commercial Tyr--Gly--Gly

Tyr--Gly--Gly is sold as a chemical reagent (L-tyrosylglycylglycine) bySigma Chemical Co., St. Louis, Mo., among others. Tyr--Gly--Gly is notsold in U.S.P. grade, and it is illegal under applicable laws to sellTyr--Gly--Gly for use as a pharmaceutical. Commercial gradeTyr--Gly--Gly is not considered free of pyrogens, endotoxin, and otherpharmaceutically unacceptable constituents. The presence of suchpyrogens, endotoxin, and other pharmaceutically unacceptableconstituents makes a product unacceptable for use as a drug, as thatterm is defined by federal statute, both under generally recognizedmedical principles and under FDA regulations. To the extent of theinventor's knowledge, no pharmaceutical preparations of this product areor have been available.

Special Terminology

As used at times in this specification, YG means Tyr--Gly (also known asL-tyrosylglycine). YGG means Tyr--Gly--Gly (also known asL-tyrosylglycylglycine).

YG-material means a member of a group consisting of a set of molecularspecies wherein each molecule contains a Tyr--Gly amino acid residuesequence, and no other amino acid residues. The molecule may be in theform of a simple Tyr--Gly sequence, or the molecule may be methylated,amidified, esterified, acetylated, etc. YG-material does not includetripeptides or higher polypeptides. However, two YG-materials (e.g., twomolecules of Tyr--Gly), may be complexed together in the form:(Tyr--Gly)Zn++(Tyr--Gly), or they may be dimerized as in the form:##STR1## Such a complex or dimer is not considered a tetrapeptide, butmerely two dipeptides complexed together or dimerized.

YGG-material means a member of a group consisting of a set of molecularspecies wherein each molecule contains a Tyr--Gly--Gly amino acidresidue sequence, and no other amino acid residues. The molecule may bein the form of a simple Tyr--Gly--Gly sequence, or the molecule may bemethylated, amidified, esterified, acetylated, etc. YGG-material doesnot include dipeptides, tetrapeptides, or higher polypeptides. However,two YGG-materials (e.g., two molecules of Tyr--Gly--Gly), or YG-materialand YGG-material, may be complexed together or dimerized. Such a complexor dimer is not considered a pentapeptide or hexapeptide.

Inhibited YG-material means YG-material that has been mixed, complexed,bound, linked, or otherwise combined with a means for inhibitingcleavage of the Tyr--Gly bond of the molecule by endogenous enzymes;however, the material must still contain a Tyr--Gly amino acid residuesequence and no other amino acid residue sequence. Puromycin andbacitracin are examples of inhibitors that may be mixed withYG-material. It is also known to N-methylate the Tyr residue to inhibitenzymatic action. It is also known to esterify or amidify the C-terminalcarboxyl group to inhibit enzymatic cleavage. The products of suchexpedients are hereinafter termed inhibited YG-material. InhibitedYG-material does not include expanded YG-material, as defined below; thetwo terms are mutually exclusive, as defined herein.

Expanded YG-material means a molecule of the form Tyr--X--Gly, where Xis a D-aminoacid, such as D-Ala. The term includes amides, esters,salts, etc., as in the case of YG-material. It is known that theinsertion of a D-aminoacid into Tyr--Gly tends to inhibit cleavage ofthe Tyr--Gly bond by endogenous enzymes. The terms YG-material andexpanded YG-material are mutually exclusive, since the former is adipeptide and the latter is a tripeptide; also the former has a Tyr--Glybond and the latter does not.

Endogenous YG-material means YG-material produced within the body.Endogenous YGG-material means YGG-material produced within the body.

Extraneous-peptide amino acid residue sequences means any and all aminoacid residue sequences except Tyr--Gly and Tyr--Gly--Gly. As usedherein, "sequence" refers to a plurality of residues, and the termsexcludes a molecule with only a single amino acid residue, such asglycine.

The abbreviations u, n, p, and f refer, respectively, to micro, nano,pico, and femto. The abbreviation M means moles or Molar, as the contextindicates; thus, fM may mean femtomoles or femtoMolar, depending oncontext.

The term "fg/kg," used in the claims in connection with a dosage amountfor a person, means femtograms of dosage material per kilogram of theperson's bodyweight. Similarly, "moles/kg" means moles of dosagematerial per kilogram of the person's bodyweight; "fM/kg" meansfemtomoles per kilogram of the person's bodyweight.

SUMMARY OF THE PRESENT INVENTION

The inventor has discovered that administration to AIDS and ARC patientsof endogenous materials containing the amino acid residue sequence YGcan alleviate certain symptoms of AIDS and ARC, can reverse certainpathological effects associated with AIDS and ARC, and appears toimprove the clinical condition of some AIDS and ARC patients. Suchtreatment does not cure AIDS and ARC, but it is therapeutically usefulin slowing the normal progression of AIDS and ARC. In particular, thetreatment significantly delays the normal progression to AIDS thatoccurs in ARC patients.

The inventor has also discovered more general immunological utility forthe foregoing endogenous materials and related synthetic materials. Thematerials of the invention are amplifiers of the immune system (as thatterm is used in Gottlieb U.S. Pat. No. 4,616,079). Thus they may be usedfor the therapeutic purposes described in the above-cited Gottliebpatents.

Generally speaking, the endogenous products of this invention are theresult of the inventor's discovery of processes leading to highlypurified forms of amplifier materials, while the synthetic products areproducts whose potential immunological utility was suggested to theinventor by his discovery of the molecular structure of immunologicallyactive constituents of the foregoing endogenous products of thisinvention. The instant products are thus more free of extraneousmaterial than any earlier amplifier products were. Further, thesynthetic instant products have the considerable advantage that they arenot derived from human body components, which are (correctly orincorrectly) thought by many persons to harbor viruses and otherpathogens.

Further, the elimination of extraneous material and the fractionation ofmixtures of molecular species into the constituent species has resultedin achieving significantly greater potency and repeatability in theresulting products than was possessed by the prior materials.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Section I of the specification describes HPLC processes for purificationof endogenous amplifier materials derived from human leukocytedialysates. Section II describes human biological assays of theimmunological activity of such materials. Section III describes humantherapeutic use of such materials, including treatment of over 30 AIDSand ARC patients. Section IV describes in vivo and in vitro tests ofsynthetic amplifiers whose molecular structure is based on that of theendogenous amplifier materials described in the preceding sections.

I. Reverse Phase Liquid Chromatography Processes

Processes are now described by which endogenous amplifier materials areextracted from human leukocyte dialysates. The procedures and reagentsused herein were chosen to provide sterile and non-toxic products forhuman treatment. The HPLC equipment used is that described in GottliebU.S. Pat. No. 4,616,079 (of which this is a continuation-in-part), cols.5-6.

EXAMPLE 1 Extraction of Beta and Zeta

Leukocyte pellets were prepared in accordance with Example 1 of GottliebU.S. Pat. No. 4,616,079 and set aside.

An aqueous potassium phosphate solution was prepared by adding 5M KOHaqueous solution dropwise to 0.02M reagent grade phosphoric acid aqueoussolution until the pH of the solution was adjusted to pH 5.0. Thesolution was delivered to a Perkin-Elmer HPLC machine, along with HPLCgrade acetonitrile. The machine was programmed to deliver the followinginput solvent gradients: (1) 10 minutes of 0.1% concentration ofacetonitrile in phosphate solution, constant gradient; and (2) 45minutes of 0.1% to 10.0%, linear gradient. The flow rate was set at 1ml/min.

Then, the leukocyte pellets were reconstituted and 5-10 mg of thematerial was loaded into an O.S. column. HPLC was commenced.

As effluent was collected, the ultraviolet absorption of the effluentwas scanned with the machine's ultraviolet detector (210 nm, fullscale=0.32 units). A plot of the absorption data of this process isshown in FIG. 1 of Gottlieb U.S. Pat. No. 4,616,079. Apparent solventconcentrations, retention times, and ultraviolet absorption wererecorded. The results of the run are summarized below:

Alpha

A distinct doublet ultraviolet absorption peak was observed at retentiontime 12-14 minutes (approximately 0.5 to 0.9% acetonitrile concentrationas observed on the machine's display and approximately 0.1 to 0.2%estimated actual concentration). The material accompanying this peak isdesignated herein as Alpha. It has no known immunological activity. Butits elution serves as an indication ("marker") that material designatedherein as Beta, which has been discovered to have amplifier activity, isabout to elute.

Beta

Beta eluted approximately 3 minutes later, between approximately 15 and20 minutes retention time. It was accompanied by a sharp, singleultraviolet absorption peak (hereinafter referred to as the Beta peak)reaching full scale. Displayed solvent concentration was 1.2 to 2.2%;estimated solvent concentration was 0.4 to 1.5%.

Frequently, in runs of this process, the material at the front edge ofthe Beta peak contained a material hereinafter described as Beta 1.12and identified as endogenous YGG-material. Sometimes, such materialappeared as a shoulder on the Beta peak.

Gamma

Immediately after Beta, at retention time between approximately 17 and22 minutes, material designated herein as Gamma eluted. The Gammamaterial was characterized by either a distinct broad peak ofultraviolet absorption or as a shoulder on the absorption indication atthe end of the elution of Beta. Gamma is without known immunologicalactivity.

Delta

A group of peaks were then observed in the retention time range of from23 to 36 minutes. The first of these peaks (a rather low one, less than30 or 40% of full scale) corresponded to (i.e., occurred with theelution of) biologic material designated herein as Delta, which has beendiscovered to have amplifier activity. Delta eluted in the retentiontime range of approximately 26-28 minutes. Solvent concentrationindicated on the visual display of the machine was from 3.5 to 4.0%,while estimated actual solvent concentration was 2.8 to 3.3%. Delta didnot maintain a well-fixed location in this process, and sometimes eitherdid not come off the column at all or was buried in the next material(Epsilon).

Epsilon

The next large absorption peak in the group was approximately 20% (ormuch more) of full scale, occurred about a minute later at retentiontime 27-30 minutes, and was a broad single peak or a doublet; the peakaccompanied material designated herein as Epsilon. Epsilon is withoutknown immunological activity.

Zeta

The absorption peak immediately following, approximately 2 minuteslater, at retention time about 29-32 minutes, is designated herein asZeta. Zeta has been discovered to have amplifier activity. As indicatedbelow, Zeta can by a further process be separated into two moieties,Zeta-1 and Zeta-2, the second of which is has been discovered to containthe entire amplifier activity of this material.

Other materials, not discussed herein, but discussed in Gottlieb U.S.Pat. No. 4,616,079, eluted thereafter in this process.

The materials of Example 1 are contaminated with phosphate ions, areimperfectly purified from extraneous material (material having no knownuseful immunological activity), and are considered unsuitable foradministration to human subjects. A further HPLC procedure with adifferent solvent system has been discovered to remove phosphate andextraneous material. The resulting material appears to be considerablymore free of extraneous material and was considered suitable foradministration to human subjects (as discussed below).

EXAMPLE 2 Beta-1.0 Process

The material of preceding Example 1 was further purified and separatedby HPLC on the analytic column. First, a 0.1% (v/v) aqueoustrifluoroacetic acid (Mallinckrodt, Inc., Paris, Ky.) solution wasprepared, and the pH of the solution was adjusted to pH 2.5 by thedropwise addition of sufficient 5M KOH aqueous solution. The solutionwas delivered to the Perkin Elmer machine, along with HPLC gradeacetonitrile.

The machine was programmed for a 45 minute linear gradient of 0.1% to45% concentration of acetonitrile in the trifluoracetic acid solution.(A 25-minute run to 25% is acceptable, but 45 minutes to 45% is moreconservative.) The flow rate was set at 1 ml/min.

Beta fractions from approximately 4 procedures of Example 1 were pooledand loaded as starter material into an O.S. column; and HPLC wascommenced. The effluents were scanned with the ultraviolet absorptiondetector, as in the preceding example. Full scale was set at 1.28absorption units.

Contaminating Gamma material eluted at 8-11 minutes and was discarded.Displayed solvent concentration range was 8-1%; estimated actual solventconcentration was 4.8-7.8%. The absorption peak associated with Gammawas approximately 30-40% of full scale.

Material hereinafter designated as Beta-1.0 eluted at 15-18 minutes.Displayed solvent concentration range was 15-18%; estimated actualsolvent concentration was 11.8-14.8%. The absorption peak was at leastfull scale and was quite sharp.

Analysis of Beta-1.0 indicates the presence of a mixture of molecularspecies. Two have been found to have intrinsic amplifier activity andare further discussed hereinafter. These two are a dipeptide materialconsisting essentially of a YG sequence, and a tripeptide materialconsisting essentially of a YGG sequence. The relative amounts ofYG-material and YGG-material vary from sample to sample, as is to beexpected with human-derived immunologically active products, but theaverage ratio of YG-material:YGG-material is between 16:1 and 20:1. Inaddition, dried Beta-1.0 contains approximately 90% phenylalanine plussmall amounts (less than 5% (w/w)) of other materials, notably Phe--Ser,Gly--Gly, and Gly--Glu; all of these materials were tested and found tolack intrinsic amplifier activity. Extensive clinical work with Beta-1.0is described hereinafter.

EXAMPLE 3 Zeta-2 Process

The procedure of Example 2 was repeated, except that the startermaterial used as Zeta material of Example 1.

Zeta fractionated into two moieties--hereinafter designated Zeta-1 andZeta-2. The first is without known immunological activity. The secondappears to have all of the amplifier activity of Zeta.

Zeta-1 eluted at approximately 11-14 minutes (displayed solventconcentration range, 11-14%; estimated actual solvent concentrationrange, 7.8-10.8%). Zeta-2 eluted at approximately 15-19 minutes(displayed solvent concentration range, 15-19%; estimated actual solventconcentration range, 11.8-15.8%). Their absorption peaks were quitesharp and were separated by an average of 2-3 minutes.

Mass spectrometer analyses of Zeta-2 did not indicate any peptidecomponents. Although not conclusive, they suggested that Zeta-2 consistsessentially of one or more molecules of the prostaglandin family, withblocked hydroxyl groups. It is believed that Zeta-2 has a M.W. ofapproximately 250 to 300.

Delta has not been recovered by use of this procedure.

Further refinements have been developed that further purify theseproducts, remove additional extraneous material, and make the materialsmore suitable for human administration. The Beta material of Example 1has been further fractionated and refined by these techniques, and theresulting products have become sufficiently purified to permit aminoacid analysis thereof. That in turn has led to a determination of theprobably molecular structure of the fractions in question.

EXAMPLE 4 Ethanol/Beta-1.1 Process

The Beta material developed from the pH 5 phosphate gradient of Example1 was dried in a vacuum evaporator without heat. The material wasreconstituted in 0.1% trifluoroacetic acid. A Perkin-Elmer preparativecolumn was used for HPLC; a 3.5 cm×28 cm column was packed withoctadecylsilane, and the reconstituted material was introduced. A flowrate of 6.0 ml/min was set. The machine was programmed to deliver aethanol-in-water linear input gradient, starting at 0% ethanol andreaching 50% at 30 minutes.

Absorbance was monitored at 254 mm, 0.1 Absorbance Units=Full Scale.Three major peaks were observed, which respectively eluted at 10.4-10.6min, 14.0-16.0 min, and 18.0-19.0 min. The biological activity wasassayed and found to reside almost entirely in the material associatedwith the middle peak, which is designated herein as Beta-1.1. The lastpeak was found to be associated with material that was mainlyphenylalanine and contained approximately 90% of the starting materialused. The first peak was lower than the second and third, in terms ofabsorbance units, and is believed to be an artifact of the systemassociated with the ethanol solvent or due to elution of salts. Thesecond and third peaks varied in relative height from preparation topreparation.

Based on refractive index measurements, the ethanol concentration of theeffluent of the second peak was found to be from approximately 0.1% toapproximately 0.4%.

An amino acid assay of the material associated with the Beta-1.1 peakwas made, with the following normalized results:

    ______________________________________                                                Asx  2                                                                        Thr  1                                                                        Ser  1                                                                        Glx  1                                                                        Pro  2                                                                        Gly  4                                                                        Ala  2                                                                        Val  1                                                                        Ile  1                                                                        Tyr  1                                                                        Lys  3                                                                        His  1                                                                        Arg  1                                                                             23                                                               ______________________________________                                    

For a number of reasons, the inventor did not believe that the Beta-1.1endogenous amplifier material was a polypeptide with 23 peptide groups,or a mixture of 3 octapeptides, or the like. The inventor determinedthat still further purification of the materials of the foregoingprocess would be desirable, to ascertain whether one or moreimmunologically active materials were present in which there weresubstantially fewer peptide groups.

To date, this has resulted in the extraction of two materials havingconsiderably greater amplifier activity per unit weight of material thanBeta-1.0 or Beta-1.1: a dipeptide material and a tripeptide material. Anacetonitrile HPLC process was developed that permitted such a result andit is described in the following example. The ZORBAX™ brand of resin(DuPont) column used hereinafter comes packed with a DuPont materialcontaining octadecylsilane groups chemically bonded to silica particles.

EXAMPLE 5 Purification of Beta-1.1 to Beta-1.11, Beta-1.12 and Beta-1.13

The Beta-1.1 material of Example 4 was injected into an O.S. HPLCcolumn, 1 ml/min flow rate, 25° C. The solvent system was 100%acetonitrile (CH₃ CN), HPLC Grade, and 0.05% trifluoroacetic acidaqueous solution, HPLC Grade, pH 2.5. The following solvent input lineargradients were used: (1) for 15 minutes, from 0-6% acetonitrile; (2) for30 minutes, from 6-40% acetonitrile.

A fraction of interest, hereinafter designated as Beta-1.11, eluted atapproximately 23.8 to 24.8 min, for a new column. It was associated witha sharp, narrow peak of UV absorption at 210 nm; this peak isapproximately the fourth absorption peak observed (some variabilityexisting). The material was lyophilized and reconstituted in normalsaline, and set aside for further use. As shown below, Beta-1.11contains endogenous YG-material.

Another fraction of interest, eluted from this material on this column,but somewhat irregularly. When it eluted, the retention time wasapproximately 22.8-23.2 min. It was associated with a distinct peak ofultraviolet absorption at 210 nm, just before the peak associated withBeta-1.11. The material was lyophilized and reconstituted in normalsaline, and set aside for further use. As shown below, this material,hereinafter referred to as Beta-1.12, contains endogenous YGG-material.

A fifth UV peak is associated with material having no demonstratedintrinsic activity. A sixth UV peak is associated with phenylalanine. Aseventh peak, discussed below, is associated with immunologically activematerial of as-yet undefined structure.

Other moieties found present in effluents of this process were Gly--Gly,Ser--Phe, and Ala, along with possibly other amino acid residues aswell. None of these other moieties appeared to possess immunologicalactivity, as assayed by DH test.

To avoid confusion between the first two moieties of interest,particularly in the case of aged columns, it has been found advantageousto run purified commercial YGG through the column as a marker. Such YGGelutes reproducibly 0.8 to 1.2 min before Beta-1.11 (approximately thesame zone as Beta-1.12).

Finally, another fraction of interest is eluted from the material onthis column, at a retention time of approximately 31.5 min. It wasassociated with a sharp peak of UV absorption at 210 nm, a seventh peakconsiderably after the third and fourth peaks associated with Beta-1.11and Beta-1.12. As discussed below, this material, hereinafter referredto as Beta-1.13, is immunologically active.

The Endogenous Amplifiers of This Invention

The Beta-1.11 material was then subjected to amino acid sequencingprocedures. Using standard techniques, such as those described inExample 4 of Gottlieb U.S. Pat. No. 4,699,898, it was determined thatBeta-1.11 contains Tyr and Gly, which come off in that order, inapproximately equal proportions, indicating the presence of aYG-material. (A protein/peptide sequencer automatically removes oneamino acid at a time from the N-terminal end of a protein or peptide fordetermination of amino acid sequence.) Some YGG-material was alsodetected in this material, which has been found to be immunologicallyactive, as is discussed below. Small amounts of Ile and Lys were alsodetected. Those may be artifacts of the procedure, or may indicate smallamounts of Ile/Lys components in a molecule or molecules present inrelatively low concentrations in the product (such as, hypothetically,Tyr--Ile--Lys, Tyr--Gly--Lys, Tyr--Gly--Gly--Ile). The inventorpresently believes that the Ile/Lys material is simply an artifact or anonsignificant component.

The Beta-1.12 material was also subjected to amino acid sequencingprocedures and was identified as containing a YGG amino acid residuesequence, indicating the presence of a YGG-material.

It has not been ascertained whether isomeric forms are present, whetherthe YG- and YGG-materials of Beta-1.11 and 1.12 have other groups boundto them (e.g., methyl, acetyl, amide), or whether complexes with metalions (such as, possibly, ZN++, Ca++, Fe++, Mn++, or Mg++) are present,and if so whether the different forms, if any, possess differentimmunological activity. While it would be desirable to ascertain this,the present state of the art makes it difficult or impossible to do so.As indicated below, certain immunological assay data suggests thatBeta-1.11 and Beta-1.12 are in some way different from chemicallymanufactured (synthetic) YG and YGG, respectively, because theendogenous products extracted from human leukocyte material appear tohave greater immunological activity than the purified synthetic peptidechemicals. (The purified chemicals do not have methyl, acetyl, etc.substitutions and they are not complexed with Zn++ or other ions,although as indicated above the endogenous products may have suchstructural variations.)

There is reason to infer that the naturally occurring Beta-1.11 and 1.12amplifier products are present in the human or animal body in the formof a complex of (X)M++(Y), where (X) and (Y) are each selected from thegroup consisting of Tyr--Gly, Tyr--Gly--Gly, or a derivative thereof,and where M++ is Zn++ or another divalent metallic ion such as Ca++,Fe++, Mn++, or Mg++. There is a negative site on the Tyr group that maybind to the M++ ion; there is also a C-terminal negative site on thelast Gly that may so bind. There may well also be a mixture of suchcomplexes.

Some work has been done on the ratio of Tyr--Gly and Tyr--Gly--Glycomponents in human materials, by radioiodinating the Tyr groups andthen separating the Tyr--Gly and Tyr--Gly--Gly moieties by gelelectrophoresis. If there were only a (Tyr--Gly)M++(Tyr--Gly--Gly)complex, one could anticipate a constant 1:1 ratio of YG and YGG. Butthat does not occur; instead, the proportions observed varied fromsample to sample, but averaged YG: YGG=18:1. Possibly, there are severaldifferent materials or complexes--such as (Tyr--Gly)M++(Tyr--Gly),(Tyr--Gly)M++(Tyr--Gly--Gly), and (Tyr--Gly--Gly)M++(Tyr+Gly--Gly) --invarying proportions, depending on as yet unidentified physiologicalparameters. Making more exact determinations has not been foundpossible, because this type of analysis is at the extreme edge of thepresent site of the art. In particular, quantities of material presentare extremely small so that accurate assay is very difficult or may beimpossible with presently available technology.

From the foregoing amino acid sequencing data, in conjunction withbiological assay data described below. The inventor concludes asfollows: Two biologically significant components of the Beta-1.1material described above may be further characterized as containing theamino acids Tyr and Gly, in a 1:1 or 1:2 ratio, and as not containingsubstantial amounts of any other amino acid. The YG- and YGG-materialspresent in the Beta materials may exist as a mixture of variableproportions; either or both molecules may be complexed with metal ions(such as Zn++, Ca++, Fe++, Mn++, or Mg++); both may be complexedtogether, with the possible addition of metal ions. The materialseliminated from the final materials, such as Phe (approximately 90% ofBeta-1.0), Phe--Set, Gly--Gly, and Gly--Gly, do not possess intrinsicimmunological activity, but they may endogenously co-act with the YG-and YGG-materials to enhance their stability or immunological activity.

The foregoing series of processes began with a leukocyte dialysatecontaining an extract from approximately 10¹⁰ leukocytes. This yieldedapproximately 84 ug of Beta-1.0 or, alternatively, somewhat lessBeta-1.1. The Beta-1.12 in turn yielded approximately 168 ng ofendogenous YG-material and 3 ng of endogenous YGG-material each. Aquantity of 1 to 5 pg of endogenous YG-material, which is approximatelyone clinical dosage unit, as discussed below, is thus derived fromapproximately 1 to 2×10⁵ leukocytes. (However, the same startingmaterial also yields about 10% of that amount of endogenousYGG-material.)

The inventor has also discovered the existence of a thirdimmunologically active component in Beta-1.1, tentatively designatedherein as Beta-1.13. This component elutes from the system of Example 5at approximately 31.5 min, for a new column. It is characterized by asharp, narrow peak of UV absorption at 210 nm; this peak isapproximately the seventh absorption peak observed in the procedure ofExample 5. Beta-1.13 is tentatively believed to be a "ring" product, andnot a peptide, having a M.W. of approximately 250 to 300. It isconceivable that Beta-1.13 is merely a Zeta-2 component that is notseparated from Beta by the technology used here, , but the inventor doesnot presently so believed. Beta-1.13 has occurred in samples inapproximately the same amount as did Beta-1.12 (i.e., 5% to 10% as muchas Beta-1.11)

II. Human Biological Assay Data

Additional biological assay methods have been developed, which were notdescribed in Gottlieb U.S. Pat. No. 4,468,379, Gottlieb U.S. Pat. No.4,616,079 (of which this application is a continuation), or GottliebU.S. Pat. No. 4,699,898. The following additional assay methods have nowbeen found advantageous:

(1) antigen-induced enhanced "leukocyte inhibitory factor" ("LIF");

(2) augmented production of interleukin-2 ("IL-2"), stimulated bymitogen, antigen, or alloantigen;

(3) enhanced generation of cytotoxic cells to Raji cells (a tumor linewhich grows in culture);

(4) augmented production of gamma-interferon, stimulated by mitogen orantigen; and

(5) enhanced expression of high-density receptors for IL-2.

The inventor has found that Beta-1.0 and Zeta-2 displayed at least threeout of the five new assay criteria as well as the enhanced DH response.However, since these materials are endogenous rather than synthetic,they vary in biological activity from time to time and depending ondonor population.

The LIF assay is described in Gottlieb et al., Modulation of Human TCell Production of Migration Inhibitory Lymphokines, J. Immunology 132:256-260 (Jan. 1984), at p. 257. Protocols for the other assays arebelieved to be known to those skilled in this art. A convenientreference for the gamma interferon assay is Sinha et al., Bio/Technology6:810 (1988) at pp. 814-15.

As stated above, it has been shown that materials of the precedingexamples have amplifier activity. The following examples illustratethis.

EXAMPLE 6 DH Assay of Beta-1.0

Serial dilutions of Beta-1.0 of preceding Example 2 were made from asolution containing the amplifier material derived from approximately4×10⁸ buffy coat leukocytes in 1 ml of aqueous saline solution.

Tetanus toxoid was selected as the antigen to challenge the immunesystem of the patient (an adult male). To 0.05 ml of tetanus toxoid,fluid diluted to 1.10 to 1/40 so as to elicit a small (preferablyslightly less than 5×5 mm) skin reaction from the patient, 0.1 ml of thediluted Beta-1.0 preparation was added. The patient was subcutaneouslyinjected with several different dilutions of Beta-1.0, and also with anequal quantity of tetanus toxoid (TT) without any Beta-1.0 addedthereto. Two approximately perpendicular diameters of each respondingskin site on the man's arm were measured at the times indicated below.("TT+₋₋ " refers to TT and a dilution of Beta to the concentrationindicated; "TT" alone is TT without Beta-1.0.)

At 5 hours, the respective responses to TT+10⁻⁸, TT+10⁻⁹, and TT were14×14 mm, 19×14, and 3×3. At 24 hours: 20×24 mm, 19×23, 14×12. Otherdilutions of Beta-1.0 produced less response.

EXAMPLE 7 DH Assay of Beta-1.11

Example 6 was repeated with serial dilutions of Beta-1.11 (endogenousYG-material), beginning with 1 microgram per microliter (ug/ul) of theproduct of Example 5. It is estimated that 168 ng of the product ofExample 5 is the amount of Beta-1.11 that can be derived fromapproximately 1×10¹⁰ buffy coat leukocytes. It is also estimated thatthe M.W. of Beta-1.11 is approximately 239. Hence, 1 ug/ul isapproximately a 4 mM solution. Dilutions to 4 fM and 0.4 fM wereprepared.

After following the procedure described in Example 6, it was observedthat at 5 hours, the respective responses to TT+0.1 ml 4 fM, TT+0.1 ml0.4 fM, and TT were 7×8 mm, 9×9, and 3×5. At 24 hours: 18×21 mm, 19×19,14×15. Other dilutions of Beta-1.11 produced less response.

EXAMPLE 7A Second Beta-1.11 DH Assay

Example 7 was repeated with serial dilutions of Beta-1.11 (endogenousYG-material), beginning with 53 nM solution of the product of Example 5.Serial dilutions of 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ were prepared; a salinecontrol was also used. TT was diluted 1/20. Erythema was measured(mm×mm) and induration was scored on a scale of 0 to +++. The followingdata were observed (and are published in Sinha et al., Biotech 6:810-15(1988), at p. 812):

    ______________________________________                                               Hours Post Injection                                                          7 Hrs         12 Hrs                                                   Dilution Erythema   Indur.   Erythema Indur.                                  ______________________________________                                        10.sup.-6                                                                              14 × 10                                                                            0        18 × 18                                                                          0                                       10.sup.-7                                                                              12 × 15                                                                            +        15 × 25                                                                          0                                       10.sup.-8                                                                              12 × 17                                                                            ++       20 × 22                                                                          0                                       10.sup.-9                                                                              10 × 12                                                                            +        20 × 21                                                                          ±                                    Saline Ctl.                                                                            4 × 5                                                                              0        10 × 10                                                                          0                                       ______________________________________                                    

EXAMPLE 8 DH Assay of Beta-1.12

Example 6 was repeated with serial dilutions of Beta-1.12 (endogenousYGG-material), beginning with 1 ug/ul of the product of Example 5. It isestimated that 3 ng of the product of Example 5 is the amount ofBeta-1.12 that can be derived from approximately 1×10¹⁰ buffy coatleukocytes. It is also estimated that the M.W. of Beta-1.12 isapproximately 295. Hence, 1 ug/ul is approximately a 3 mM solution.Dilutions to 3 fM and 0.3 fM were prepared.

After following the procedure described in Example 6, it was observedthat at 5 hours, the respective responses to TT+0.1 ml 3 fM, TT+0.1 ml0.3 fM, and TT were 7×8 mm, 9×9, and 3×5. At 24 hours: 18×21 mm, 19×19,14×15. Other dilutions of Beta-1.12 produced less response.

EXAMPLE 8A Second Beta-1.12 DH Assay

Example 8 was repeated with serial dilutions of Beta-1.12 (endogenousYGG-material), beginning with 64 nM solution of the product of Example5. Serial dilutions of 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ were prepared; twosaline controls were also used. TT was diluted 1/80. Erythema wasmeasured (mm×mm) and induration was scored on a scale of 0 to +++. Thefollowing data were observed (and are published in Sinha et al.,Bio/Technology 6:810-15 (1988), at p. 812l):

    ______________________________________                                               Hours Post Injection                                                          7 Hrs         12 Hrs                                                   Dilution Erythema   Indur.   Erythema Indur.                                  ______________________________________                                        10.sup.-6                                                                              2 × 2                                                                              0        0        0                                       10.sup.-7                                                                               9 × 10                                                                            +        16 × 16                                                                          +                                       10.sup.-8                                                                              12 × 13                                                                            ++       18 × 21                                                                          ++                                      10.sup.-9                                                                              10 × 13                                                                            ++       16 × 17                                                                          +                                       L Saline Ctl.                                                                           8 × 10                                                                            +        16 × 17                                                                          +                                       R Saline Ctl.                                                                          8 × 8                                                                              +        16 × 17                                                                          +                                       ______________________________________                                    

EXAMPLE 8B DH Assay of Beta-1.13

Example 6 was repeated with serial dilutions of Beta-1.13 (thirdendogenous material), beginning with 1 ug/ul of the product of Example5. It is estimated that 3 ng of the product of Example 5 is the amountof Beta-1.13 that can be derived from approximately 1×10¹⁰ buffy coatleukocytes. It is also estimated that the M.W. of Beta-1.13 isapproximately 400. hence, 1 ug/ul is approximately a 2.5 mM solution.Dilutions to 2.5 fM and 0.25 fM were prepared.

After following the procedure described in Example 6, it was observedthat at 7 hours, the respective responses to TT+0.1 ml 2.5 fM, TT+0.1 ml0.25 fM, and TT were 11×17 mm, 10×16, and 9×9. At 24 hours: 13×12 mm,14×12, 6×9. Other dilutions of Beta-1.13 produced less response.

EXAMPLE 9 Effect of Beta-1.11 on T-helper Cell IL-2 Receptor Expression

Serial dilutions of Beta-1.11 were prepared, beginning with a 81.3 nMconcentration and then serially diluting that further by factors of1000, 2000, 4000 . . . 512,000. The 1/512,000 dilution produces apreparation that is a 159 fM concentration of Beta-1.11.

A tetanus toxoid (TT) preparation was prepared of strength 0.1flocculation unit (L_(f)) per ml.

Cell cultures were prepared with the TT preparation alone, and with theTT preparation combined with the serial dilutions of Beta-1.11. Thecultures were incubated and mixed with a suitable antibody against IL-2receptors. Data for receptor expression was tabulated both for T-helpercells bearing a low density of receptors and for T-helper cells bearinga high density of receptors. (The T-helper cells bearing a high densityof receptors are considered to be the ones that are immunologicallyactive.) The cells were from normal subjects.

In the case of the TT preparation alone, 4.7% of T-helper cellsexpressed a low density of IL-2 receptors and 0.40% of T-helper cellsexpressed a high density of IL-2 receptors.

There was a plateau of maximal expression for T-helper cells bearing alow density of receptors from TT+20.3 pM Beta-1.11 (9.9% expression) toTT+1.3 pM (8.4%). The highest figure for high density of receptors wasat 159 fM (9.9%). This highest figure occurred at a point where therewas a steadily ascending curve. Accordingly, it si believed that iffurther dilutions of Beta-1.11 had been used a still higher figure wouldhave been reached, before the expression percentage began to drop. It isnot possible to state, from this data, where the maximum would occur,but it seems likely to be somewhere between 80 fM and 20 fM.

The low density plateau reflected an approximately doubled expressionrate of IL-2 receptors as a result of Beta-1.11. The high densitymaximum, which the inventor considers more relevant, reflected anincrease of IL-2 receptors by a factor of approximately 25 as a resultof Beta-1.11, and that figure probably falls short of the increase thatwould result from using still more diluted Beta-1.11.

EXAMPLE 10 Effect of Beta-1.11 on Antigen-Induced Production of GammaInterferon

Preparations of 0.1 L_(f) /ml and 1.0 L_(f) /ml TT were used todetermined the effect of Beta-1.11 (endogenous YG-material) onproduction of gamma-interferon in normal cells. As before, serialdilutions of Beta-1.11 were prepared from 46.5 pM to 91 fM.

The 0.1 L_(f) /ml and 1.0 L_(f) /ml TT preparations, alone, respectivelyinduced baseline production of 1.0 and 10.4 units/ml ofgamma-interferon.

0.1 L_(f) /ml TT+317 fM Beta-1.11 produced 29.8 units/ml, the observedmaximum, and 0.1 L_(f) /ml TT+159 fM Beta-1.11 produced 29.1 units/ml.

1.0 L_(f) /ml TT+159 fM produced 25.6 units/ml, the maximum.

In the case of the 0.1 L_(f) /ml TT preparation, the maximum wasapproximately 30 times the baseline amount. In the case of the 1.0 L_(f)/ml TT preparation, the maximum was approximately 2.5 times the baselineamount.

Generally, the DH assays showed that Beta-1.11, Beta-1.12, and Beta-1.13are immunologically active and they appear to be more active (per unitweight) then Beta-1.0, in the setting of these assays. The apparentincrease in potency may be attributed to the elimination ofphenylalanine and other amino acid product, which are without intrinsicimmunological activity.

In both of the TT+Beta-1.11 in vitro immunological tests, expression ofhigh-density IL-2 receptors and antigen-induced production ofgamma-interferon, the optimal concentration of Beta-1.11 appeared to beapproximately 317 fM for gamma-interferon and approximately 159 fM forhigh-density IL-2 receptors. Since workers in this field consider thesetwo assays generally indicative of immunological effect, and since theyhave been found correlated to clinical immunological effect in the caseof other Beta materials from which Beta-1.11 is derived, the inventorconsidered that the assays indicated a very high probability thatBeta-1.11 would produce in vivo amplifier effect in human subjects. Asshown below (Examples 17-18), this prediction has been confirmed byactual clinical data. Thus, the materials of this invention have beenshown to stimulate gamma-interferon production and to enhance productionof receptors for IL-2. These are among the most important known effectsof modifiers of biological responses produced in lymphocytes.

In the case of Beta-1.12, it is not certain whether the DH response isto the Beta-1.12 per se (i.e., to material essentially consisting ofendogenous YGG-material) or to a Beta-1.11 metabolite (i.e., materialconsisting essentially of endogenous YG-material) of Beta-1.12. It isknown that pentapeptides (enkaphalins) containing a YGG amino acidresidue sequence are degraded in vivo by the action ofdipeptidylaminopeptidase, which hydrolyzes enkephalins (e.g., H₂N--Tyr--Gly--Gly--Phe--Met--OH) by cleaving the Bly--Bly amide bond.(See, e.g., Schwartz et al., op. cit. supra (Background section ofspecification), at p. 1716, citing and summarizing studies in thisfield.) Hence the apparent immunoamplificatory effect of YGG-materialmight be attributable to YG-material as a metabolite of YGG-materialrather than to YGG-material itself.

On the other hand, both of these endogenous materials may have intrinsicimmunoamplificatory effect. For example, the receptor site for thislymphokine could be associated with the Tyr--Gly portion of themolecule, and the additional Gly amino acid residue may have a neutraleffect on reception and biological activity. It is also possible thatYGG interacts with a different receptor site to have the same biologicaleffect as YG. Alternatively, a complex of both peptides, perhaps througha trace metal (such as Zn++, Ca++, Fe++, Mn++, or Mg++) may be required.This is a question calling for further work, such as in vitro tests ofTry--Gly--Gly in the presence of a suitable aminopeptidase inhibitor, toprevent cleavage of the Gly--Gly amide bond in Tyr--Gly--Gly to produceTyr--Gly. At the present, the inventor believes it proper only toconclude that the YGG data shows a potent direct or indirectimmunoamplificatory effect.

EXAMPLE 11 DH Assay of Zeta-2

The procedure of Example 6 was repeated with Zeta-2 of Example 3.However, purified protein derivative of tuberculin ("PPD") wassubstituted for TT.

At 12 hours, the respective responses to PPD+10⁻⁶, PPD+10⁻⁷, PPD+10⁻⁸,and PPD were 1×1 mm, 15×22, 14×11, and 1×1. At 27 hours: 2×2 mm, 22×24,15×15, and 3×3.

III. Human Therapy Tests of Endogenous Amplifier Materials

The effectiveness of above-described endogenous amplifier materials inamplifying human immune system response has been tested in over 100 mensuffering from AIDS or ARC. All amplifier materials used in this workwere free of endotoxin as detected by the Limulus assay (M.A.Bioproducts, Rockville, Md.). all material used was also pyrogen-free.The following examples illustrate these tests. (As used hereinafter, theterm "T-helper cell" includes cells designated as T4⁺, CD4⁺, Leu3⁺, andT4.)

The inventor has ascertained by empirical means that an effective dosageamount of these products, in the procedures described hereinafter, isthat derived from 125,000 leukocytes, purified by the method of Example2 and dispensed in 0.5 ml of normal sterile saline solution. (Thisdosage amount, that derived from 1.25×10⁵ leukocytes, is frequentlyreferred to hereinafter as "one standard dose of Beta.")

EXAMPLE 12 Multiple Doses of Beta-1.0 With Transfusion

An AIDS patient, DT, with Kaposi's sarcoma was given doses of Beta-1.0(a preparation containing YG-material, YGG-material, phenylalanine, andother materials) together with transfusions of isologous leukocytesavailable from DT's indentical twin brother (a normal, disease-freeperson). DT also received such transfusions without Beta-1.0.

An initial transfusion of approximately 1.0×10¹⁰ isologous leukocytes(without Beta-1.0) produced a moderate restoration of DT'sphytohemagglutin (PHA) proliferative. Within 13 days the responsedeclined to baseline levels with no concomitant alteration in the ratioof circulating helper leukocytes to suppressor leukocytes (T4/T8 ratio).

Ten days after the initial transfusion, DT was given a single standarddose of Beta-1.0 (as stated above, that which is derived from 400,000leukocytes). No effect was observed on DT's PHA response.

A cycle of treatment comprised of a second isologous leukocytetransfusion (again, the same number of leukocytes) followed at 24, 48,and 71 hours by subcutaneous administration of Beta-1.0 doses derivedfrom 400,000, 4,000,000, and 400,000 leukocytes, respectively (i.e.,3.2, 32, and 3.2 standard doses). A significant increase in DT's PHAresponsiveness followed. It was associated with an increase in the T4/T8ratio, resulting from an absolute increase of T4+ cells and a decreasein T8+ cell numbers. After approximately one month, these parameters ofimmune system response declined to approximately their former level.

A third transfusion similar to the first (no Beta-1,0) was given. Noeffect on PHA response or T4/T8 ratio was observed.

While these studies were made, parallel studies of IL-2 production weremade. Initially, no IL-2 production was observed in response to PHA.this correlated with the patient's low proliferative response tomitogen. The initial leukocyte transfusion did not affect thisparameter. After the second transfusion (leukocytes and Beta-1.0),significant levels of IL-2 were induced by PHA. It is considered thatthis data (and similar data in the next two examples) confirms the invitro data of Examples 9-10 and the discussion following those examples,concerning stimulation of lymphokine production.

EXAMPLE 13 Multiple Doses of Beta-1.0: Group 1

The members of a group of 15 patients with AIDS or ARC received onestandard dose of Beta-1.0 once every month until three doses were given(three months). Of these 15 patients, six had candida infections (oralcandidiasis), and 12 had Kaposi's Sarcoma.

Clinical symptoms were monitored. No decrease in weight was observed. Notoxicity to Beta-1.0 was observed.

A significant decrease in candida infection was observed as a result oftreatment, in three-quarters of the patients completing the monthly doseprotocol.

Skin test sensitivity (DH test) to tetanus toxoid was noticeablyenhanced in the test subjects, and returned to an approximately normallevel in 47% of the subjects. Since, according to the Walter ReedClassification of Severity of AIDS/ARC (see 314 new Eng. J. Med. 131(1986)), candida infection and loss of skin test sensitivity are signsof far-advanced immunodeficiency, the inventor believes that theeffectiveness of Beta-1.0 in reversing these symptoms is significant.

Mitogen-stimulated leukocyte proliferation increased with eachsuccessive dose. Mitogen-stimulated IL-2 production increased in atleast 60% of these patients. Response to pokeweed mitogen (PWM)increased for those patients having more than 50-100 T4 cells per mm³remaining.

EXAMPLE 14 Multiple doses of Beta-1.0: Group 2

The members of a group of 14 patients with AIDS or ARC received onestandard dose of Beta-1.0 every two weeks for six doses (approximatelythree months). Of these subjects, six had candida infections. Of the 14patients, 11 had Kaposi's Sarcoma.

Clinical symptoms were monitored. Eleven of the 14 patients gainedweight. An average weight gain of 4.4 lb occurred in these 11. Notoxicity to Beta-1.0 was observed. Serum uric acid levels fell. Creatinephosphokinase levels fell. Since high levels of uric acid and creatinephosphokinase reflect tissue breakdown characteristic of AIDS, theinventor believes that lowering of the levels of these substances andreversal of weight loss indicates significant clinical improvement.

Skin test sensitivity to tetanus toxoid returned in 57% of subjects.Candida infection was totally cleared in three subjects (50% of the sixwith candida infections) and decreased in another.

Mitogen-stimulated leukocyte proliferation increased. Mitogen-stimulatedIL-2 production increased in 60% of the patients after two doses ofBeta-1.0; and in all those patients having more than 50-100 T-helpercells/mm³ remaining, after two doses of Beta-1.0.

Response to pokeweed mitogen (PWM) increased for those patients havingmore than 50-100 T-helper cells/mm³ remaining. A small increase to PWMappeared after the second dose with those patients having fewer than50-100 T-helper cells/mm³ remaining, and slowly increased following thenext two doses.

There was also observed a slowing of the rate of destruction of T-helpercells in these AIDS and ARC patients during their treatment withBeta-1.0. For example, untreated patients with ARC typically loseT-helper cells at the rate of approximately 13.4 cells/month. For thoseARC patients who received Beta-1.0 on a monthly basis (Example 13), therate of T-helper cell loss was 7.2 cells/month, which is approximatelyhalf the typical rate. For those patients who received Beta-1.0 everytwo weeks (Example 14) the rate of T-helper cell loss was 4.2cells/month, which is approximately a third the typical rate. It isbelieved that this data indicates that Beta-1.0 slows the rate ofT-helper cell destruction typical of ARC, and that the retarding ofdestruction observed here was proportional here to the dosage.

EXAMPLE 15 Multiple Doses of Beta-1.0: Group 3

Five patients, three with AIDS (RB, JB, and RG) and two with ARC (WW andCM) were treated with Beta-1.0 over a period of approximately a year ormore. (One standard dose administered intradermally every two weeks.)

Skin test sensitivity returned completely in three subjects andpartially in one (RB, JB, WW, and CM). Candida infection improved in thetwo patients (RB and CM) initially having it and it did not appear inthe others. The percentage of T-helper cells increased transiently infour patients (RB, RG, WW, and CM).

Three patients gained substantial weight (RB, WW, and CM).PHA-stimulated lymphocyte proliferation increased in all five, PWMresponse in four (RB, RG, WW and CM), IL-2 production in three (RB, WW,and CM).

A sixth patient was originally included in this group, but hisimmunodeficiency was so severe on presentation that he succumbed to anoverwhelming opportunistic infection before immunological reconstitutioncould be affected.

EXAMPLE 16 AIDS/ARC Conversion in Placebo-Controlled Multicenter Testsof Beta-1.0

In order to secure clinical data for FDA licensing of beta-1.0,double-blind, randomized, placebo-controlled trials were conducted on atotal of 141 ARC patients at eight testing centers, over a six-monthperiod.

93 patients diagnosed as having ARC received one standard dose ofBeta-1.0 biweekly for 26 weeks. 48 patients diagnosed as having ARCreceived a placebo dose biweekly for 26 weeks. Attending physiciansmonitored the patients for clinical symptoms.

In particular, focus was directed to diagnosis of the conversion to AIDSin these ARC patients. This conversion is marked by a significant changein symptoms and clinical status, such as development of Pneumocytiscarinii pneumonia, tracheobronchial candidiasis, or Kaposi's Sarcoma.Such a clinical event is termed an "endpoint," since it marks the end ofARC and the beginning of AIDS, which appears to be invariably fatal, andis attended by more severe clinical symptoms.

At the end of 26 weeks, endpoints had appeared in 12 of the 48 placebopatients, representing 25% of that population; and in 4 of the 93patients given Beta-1.0, representing 4% of that population. The rate ofARC/AIDS conversion in patients treated with Beta-1.0 was thusapproximately 20% of the conversion rate in placebo patients. SinceARC/AIDS conversion is clinically highly significant, it is believedthat the foregoing trial data supports FDA licensure of Beta-1.0 for ARCpatients, to delay conversion to AIDS; and the data has been submittedto FDA for that purpose.

The patients treated with Beta-1.0 in these trials were also observed toshow improved clinical symptomatology comparable to that described abovein Examples 13-14, such as lessened weight loss, lessened fever, lesscoughing, and less diarrhea.

EXAMPLE 17 Three-month Dosage of Beta-1.11 (ARC Patient)

ARC patient A received a total of six doses of 0.1 pg of Beta-1.11(endogenous YG-material), given biweekly for approximately three months.His immune responsiveness was measured by determining the reactivity ofhis leukocytes to pokeweed mitogen (PWM).

A baseline responsiveness was established before treatment at 6241units, hereinafter designated as 100%, for comparison purposes. Hissubsequent PWM reactivity measurements are tabulated below:

    ______________________________________                                        Response after Dose No.                                                                          Units   Percent                                            ______________________________________                                        0                    6241   100%                                              (baseline)                                                                    1                  14,027  225                                                2                  13,875  222                                                3                  12,058  193                                                4                  11,753  188                                                5                  10,358  166                                                ______________________________________                                    

It was also observed that A regained his DH reaction to tetanus toxoidas a result of treatment with Beta-1.11. the foregoing data indicatedthat dosage with endogenous YG-material at least partially reversed thispatient's immunodeficiency.

The trend of observed PWM reactivity suggests that the effect of 0.1 pgwas less after several doses had been administered, and that lower dosesshould be used for subsequent treatment. That is, a dose that is optimalon starting therapy will in some instances have to be reduced as timegoes on. The inventor has observed that there is an optimal dosage ofamplifier, and that doses above or below the optimum amount produce lessamplifier effect than the optimal dose does (see, e.g. Gottlieb U.S.Pat. No. 4,468,379, col. 13-14, 16-17; copending application, Diagnosticmethods for immune function, Ser. No. 830,728, now issued as U.S. Pat.No. 4,778,750, Sections III-IV, cols. 5-7).

EXAMPLE 18 Two-month Dosage of Beta-1.11 (ARC Patient)

ARC patient B had previously been treated with a series of standarddoses of Beta-1.0 (a preparation containing endogenous YG-material,endogenous YGG-material, phenylalanine, and other material) toreconstitute his immune system. He was then given biweekly doses of 0.1pg of Beta-1.11 (endogenous YG-material) for a total of four doses.

His immune function was measured by determining PWM reactivity, as inthe case of Patient A. His reactivity throughout remained at slightlybelow normal levels. His DH response to tetanus toxoid also remained atslightly below normal levels. The foregoing data indicate that dosagewith endogenous YG-material maintains in effect the restoration ofimmunodeficiency brought about by dosage with Beta-1.0.

The data for Patient B suggest that 0.1 pg was approximately the correctdosage amount for this patient (i.e., an effective dosage amount).

The foregoing examples indicate that endogenous YG-material (Beta-1.11)and materials containing it (such as Beta-1.0) stimulate the T-helpercell population of the human body. This suggests that Beta materials areuseful in improving human immune response characterized by a T-helpercell defect, by amplifying immune responsiveness. It is believed thatdoses of Beta partially restore the functioning of a defective subset ofthe T-helper lymphocytes. Tests such as that conducted on patient DTsuggest that Beta can partially correct a defect in T-helper cellfunction even in the presence of the excessive proportions of T8+ cellsobserved in AIDS patients. It appears, further, that some minimal levelof residual T-helper cell function must be present for Beta to amplifyand thus improve immunological functions; if T-helper cell loss is toosevere, there may not be enough T-helper cells left to respond to dosesof Beta as a lymphokine and thus be immunologically reconstituted. Thedata above suggest that when the total T-helper cell population fallsbelow approximately 100 cells/mm³, it is difficult or impossible toreconstitute immunological function.

Generally speaking, 0.5 to 5.0 pg of Beta-1.1 (endogenous YG-material)or 0.5 to 5 ng of Beta-1.0 (as defined by phenylalanine content),administered biweekly by intradermal or subcutaneous injection, are eacheffective dosage amounts for adult male (70 kg) AIDS or ARC patients.Those dosages are approximately equivalent to 7 to 70 fg/kg of YG and 7to 75 pg/kg of Beta-1.0 (based on phenylalanine content), respectively.Because of patient-to-patient individual variations, the difference invarious immuno-deficient clinical conditions, and the need to allowexercise of professional judgment by the attending physician, thesedosage amounts must be considered subject to change by a factor ob 5 to10 in each direction. That translates to a dosage range of approximatelyfrom 0.5 to 500 fg/kg of YG, and from 3×10⁻¹⁸ to 3 f moles/kg of YG(using a GMW of YG=239).

The above-described clinical work has primarily been that conducted withIMREG-1, which has been found to be a mixture of Beta-1.11 (endogenousYG-material), Beta-1.12 (endogenous YGG-material), Beta-1.13,phenylalanine, Phe--Ser, Gly--Gly, Gly--Glu, and amounts of some otheramino acid products. It is generally believed desirable to administer astandardized product to patients, rather than a variable biologicalproduct whose constituents differ from a dose to dose depending on thesource material. Thus, the inventor considers that it would probably bepreferable to administer a predetermined mixture of Beta-1.11(endogenous YG-material), Beta-1.12 (endogenous YGG-material),Beta-1.13, and perhaps other material, rather than unstandardizedIMREG-1, subject of course to FDA regulatory constraints.

This suggests a preferred procedure for using these endogenousmaterials: Purification to the Beta-1.11, Beta-1.12, and Beta-1.13 levelis carried out. That is followed by remixture in accordance with apredetermined formula. As indicated elsewhere, the average ratio ofYG:YGG:Beta-1.13 in IMREG-1 has been found to be approximately 18:1:1.If, as may be the case, the three amplifiers must be concurrentlypresent for optimal biological results, the predetermined mixture shouldcontain them in approximately that ratio (approximately 90%/5%/5%/). Theinventor considers that this ratio should be subject to a range of fromapproximately 10:1:1 to 25:1:1, paralleling natural variations in theproportion of materials in different human samples.

It is as yet not ascertained whether the phenylalanine, Phe--Ser,Gly--Gly, Gly--Glu, and amounts of some other amino acid products whichhave been found to be present in IMREG-1 contribute anything toimmunological activity. The inventor has shown that these componentslack detectable intrinsic immunological activity. However, that does notrule out the possibility that they may act naturally as stabilizers,preventers of hydrolysis, or otherwise as adjuncts of the intrinsicallyactive components of IMREG-1. Therefore, clinical work may show that thepredetermined mixture discussed in the preceding several paragraphsshould contain some of these components, which were actually present inthe product (IMREG-1) primarily used in clinical tests with AIDS and ARCpatients. Hence, the predetermined mixture may appropriately includephenylalanine, Phe--Ser, Gly--Gly, and Gly--Glu, found in the natural(endogenous) product. This would provide a standardized product and yetone more closely paralleling the composition of the IMREG-1 materialused in large scale clinical tests with AIDS and ARC patients.

The following present-tense example is intended to illustrate andsummarize the teachings of the preceding examples describing actualclinical data with over 100 AIDS and ARC patients.

EXAMPLE 19 Increase of Immune Response

A patient is known to suffer from immunodeficiency. The immunodeficiencyis attributed to AIDS, ARC, chemotherapy, radiation treatment, oranother known cause of immunodeficiency.

Endogenous amplifiers are prepared and purified as described in SectionI, supra, and are pooled, lyophilized, and redissolved in normal salineor other physiologically acceptable vehicles. An effective dose (e.g.,0.1 ml containing the amount of material extracted from 125,000leukocytes) is injected intradermally. Increased immune responsivenessoccurs and is monitored by the patient's reactivity to an antigen towhich he is known to be sensitive (e.g., tetanus toxoid), comparingreactivity before and after administering the amplifiers.

Amplifiers are further administered either individually, or incombination, depending upon the desired effects. The persistence of thesystemic modulation produced by administration of the amplifiers variesfrom patient to patient, and must therefore be monitored periodicallywith a suitable sensitivity test, e.g., by DH assay as described above.

Additional doses are administered as required to maintain a desiredamplification of immunity, based upon the professional judgment of theattending physician. That is, the dosage should be increased over thatspecified above, if immune responsiveness is insufficient (but not tothe level where increasing doses decrease immune response); and thedosage should be decreased from that specified above, if immuneresponsiveness is greater than that which the physician considersappropriate.

As indicated in Example 6, Zeta-2 has immunoamplifier effect similar tothat of Beta-1.0, although apparently slower in terms of onset ofeffect. In addition to increasing DH response, Zeta-2 was found toincrease gamma interferon production and to enhance expression of IL-2receptors, in vitro. From the foregoing in vivo and in vitro data, whichthose persons familiar with this field generally regard as importantpredictors of clinical immune response, the inventor concluded thatZeta-2 possesses immunoamplifier activity similar to that of the Betagroup.

EXAMPLE 19A Zeta-2 Immune Response

The procedure of Example 19 is repeated with another patient known tosuffer from immunodeficiency. An effective dose (0.1 ml sterile salinecontaining the amount of material extracted from 125,000 leukocytes,tested for freedom from endotoxin and pyrogen) is injectedintradermally, biweekly for 12 weeks.

The patient's reactivity to PPD (DH assay) increases. Mitogen-stimulatedlymphocyte proliferation progressively increases with additionaldosages. Mitogen-stimulated IL-2 production also increases.

The patient is retested six weeks after conclusion of the 12-weekcourse. Reactivity to PPD, mitogen-stimulated lymphocyte proliferation,and mitogen-stimulated IL-2 production are found to be at approximatelythe initial (week-0) level.

It is concluded that administration of Zeta-2 increases immuneresponsiveness in the patient.

The above-described discovery that human leukocyte dialysates containendogenous amplifier materials, which can be extracted, purified, andadministered to patients, is considered significant in several respects.First, it significant that these materials may be isolated from normalindividuals, rather than from specific identified donors, because thispermits (and in fact has actually permitted the inventor's assignee toengage in) large-scale purification of these materials from pooled humansources. Second, the discovery that some endogenous amplifier materialcontains as intrinsically active components low-M.W. peptide productsopens the way to basically chemical, rather than basically biological,preparation of amplifier compositions. This matter is discussed in thefollowing section of the specification. Furthermore, as described inmore detail below, this discovery also paves the way to molecularmanipulation of the products already discovered, in order to improvetheir pharmaceutical properties.

IV. In Vitro and in Vivo Tests of Synthetic Amplifiers A. Synthetic YG

The endogenous product designated above as Beta-1.11 has been shown, byamino-acid analysis, to consist essentially of YG-material. It is notnow known, and may not be ascertainable in the present state of the art,whether the YG of endogenous YG-material exists as a simple YG moleculeor as a more complex molecule, such as an ester, methylate, acetylate,salt, and so on.

As indicated in the Background section of this specification, YG existsas a commercial synthetic product. The inventor has discovered a processfor purifying it to U.S.P. grade or equivalent, and has discovered thatthe product possesses intrinsic amplifier activity. In the followingsection of this specification, in vitro and in vivo assay data isdescribed for synthetic YG. All references to commercial grade YG are tocommercially available L-tyrosylslycine (Sigma Chem. Co., St. Louis,Mo.). Commercial YG is not sold in U.S.P. grade, and it is not lawful tosell the product as a drug (pharmaceutical), as "drug" is defined byfederal law. Commercial grade YG contains pyrogens and endotoxin, amongother medically unacceptable components. The following procedure makesavailable for the first time a L-tyrosylglycine product free ofpyrogens, endotoxin, and other medically unacceptable contaminants.

EXAMPLE 20 Purification of YG

Commercial grade YG was purified of pharmaceutically unacceptableconstituents or contaminants by the following procedure: 1 ug of YG wasdissolved in 1 ul of normal saline, and was then added to 99 ul of 0.05%trifluoroacetic acid aqueous solution.

50 ul of the resulting solution was injected into a reverse-phasehigh-pressure liquid chromatography analytic column (ODS AnalyticColumn, DuPont ZORBAX™, 4.6 mm×25 cm). The column was eluted with thefollowing combination of linear gradients, using HPLC grade 100%acetonitrile against 0.05% trifluoroacetic acid aqueous solution assolvent, 1 ml/min flow rate: (1) from 0 min to 15 min, 0% to 6%acetonitrile concentration; (2) from 15 min to 45 min, 6% to 40%acetonitrile concentration.

Absorbance was monitored at 210 nm, 0.05 O.D. units full scale. A sharp,narrow peak occurred at approximately 16.0 to 17.0 min, much higher thanany other peaks. The elutant accompanying the peak was purified YG,substantially free of contaminants. Specifically, the material was foundto be free of endotoxin and pyrogen.

EXAMPLE 21 DH Assay of YG/YGG

Purified synthetic YG of Example 20 and purified synthetic YGG ofExample 1 of Gottlieb U.S. Pat. No. 4,699,898 were dissolved intosterile saline, in a 2:1 ratio, to concentrations, respectively, of 220nM and 110 nM. (this mixture had an effective amplifier concentration of330 nM.) Further 1:10 successive dilutions of this mixture into sterilesaline were prepared, to provide mixtures containing 220 fM, 22 fM, 2.2fM, and 0.22 fM concentrations of YG (and corresponding molarconcentrations of YGG and YG/YGG as indicated above).

Samples of 0.1 ml of each dilution were injected intradermally into anormal human volunteer, a 70 kg adult male, in combination with a knownrecall antigen--tetanus toxoid (TT), 0.05 ml, 1/40 dilution of standard(Squibb/Connaught). As a control, the same amount of TT was injectedwith 0.1 ml normal sterile saline (0 fM YG).

The following lesions were observed (mm×mm) at the times indicated:

    ______________________________________                                        YG Conc.                                                                              220 fM   22 fM    2.2 fM 0.22 fM                                                                              0 fM                                  ______________________________________                                         6 hr.  7 × 6                                                                            7 × 8                                                                             7 × 5                                                                         4 × 4                                                                          7 × 4                           12 hr.  6 × 5                                                                            13 × 13                                                                          10 × 6                                                                         8 × 5                                                                          6 × 5                           24 hr.  3 × 4                                                                            10 × 9                                                                           10 × 9                                                                         7 × 7                                                                          3 × 2                           ______________________________________                                    

EXAMPLE 21A Another DH Assay of YG/YGG

The procedure of Example 21 was repeated five weeks later, using thesame normal test subject and the same reagents. The purpose was to shownormal human variability in DH response as modulated by theseamplifiers, in a single subject over time.

The following lesions were observed (mm×mm) at the times indicated:

    ______________________________________                                        YG Conc.                                                                              220 fM   22 fM    2.2 fM 0.22 fM                                                                              0 fM                                  ______________________________________                                         6 hr.  6 × 5                                                                            7 × 8                                                                            6 × 5                                                                          3 × 4                                                                          5 × 5                           12 hr.  12 × 13                                                                          16 × 16                                                                          17 × 14                                                                        15 × 12                                                                        12 × 12                         24 hr.  4 × 4                                                                            3 × 3                                                                            6 × 6                                                                          4 × 4                                                                          6 × 9                           ______________________________________                                    

EXAMPLE 22 Tyr+Gly DH Assay

As another control, the procedure of Example 21 was carried out with amixture of the single amino acids Tyr and Gly, in place of the mixtureof YG and YGG used in Examples 21 and 21A. The same amount of TT wasused. This test was carried out two weeks before Example 21, on the samesubject.

The following lesions were observed (mm×mm) at the times indicated:

    ______________________________________                                        T + G                                                                         Conc.   220 fM   22 fM    2.2 fM 0.22 fM                                                                              0 fM                                  ______________________________________                                         6 hr.  0 × 0                                                                            0 × 0                                                                            0 × 0                                                                           0 × 0                                                                         0 × 0                           12 hr.  6 × 5                                                                            5 × 4                                                                            4 × 4                                                                           3 × 3                                                                         3 × 3                           24 hr.  11 × 10                                                                          12 × 13                                                                          12 × 13                                                                        10 × 9                                                                         10 × 11                         ______________________________________                                    

In addition, reference should be made to the previously cited paper in 6Bio/Technology 810 (1988). In Table 3 of the paper, at p. 814, the datacomparing the immunological activity of synthetic YG (referred to as B4)with that of endogenous YG-material (referred to as ZB-4) indicated thatendogenous YG-material was 10 to 20 times as active as synthetic YG ininducing formation in vitro of gamma interferon. This data supersedessome earlier and more limited data of the inventor in which it appearedthat synthetic YG was slightly more potent (by about half an order ofmagnitude) than endogenous YG-material in inducing formation in vitro ofgamma interferon.

It is noted that in Examples 21 and 21A, using a synthetic YG/YGGmixture, amplification of DH response occurred in the range of 2.2 fM to22 fM YG (3.3 fM to 33 fM total amplifier). This may be compared withthe assays of Examples 6-8, in which DH tests were performed withBeta-1.0 (mixture of endogenous YG-material and YGG-material, withextraneous material present), Beta-1.11 (endogenous YG-material), andBeta-1.12 (endogenous YGG-material). These tests appear to indicate agreater apparent potency on the part of the endogenous products. Nosignificant effect on DH response to TT occurred with the mixture ofsingle amino acids.

Strictly speaking, the results of the DH assay of synthetic YG/YGG ofExamples 21-21A cannot be compared precisely with the DH assays ofendogenous YG-material and endogenous YGG-material of Examples 7-8,because different human test subjects were used in the tests ofsynthetic and endogenous materials. The results of these four assays,however, are consistent with the inventor's empirical observations that,speaking very, very approximately, the synthetic amplifier compounds YGand YGG are an order of magnitude less potent than the correspondingendogenous amplifier materials Beta-1.11 and Beta-1.12. At this time,there is not what the inventor considers a proper scientific basis onwhich to explain the seeming difference. However, it is clear that thematerials cannot be identical, given this observed disparity inactivity. Some possible hypotheses to account for the disparity arestated below, at the end of this section of the specification.

EXAMPLE 23 T-helper Cell Receptor Expression Assay of YG

The T-helper cell receptor expression assay of Example 9 was repeatedwith synthetic YG of Example 20.

The highest figure for high-density of receptors was at approximately 22fM, and reflected an increase of IL-2 receptors by a factor ofapproximately 1.6 as a result of YG. (This may be compared with Example9 in which endogenous YG-material was most active at approximately thesame molar concentration, but increased receptors by a factor ofapproximately 25.)

EXAMPLE 24 Mouse Tests of YG

Ten BDF₁ strain mice are divided into two groups of 5. Each mouse isinjected with 100 L1210 leukemia cells from ascitic fluid of DBA/2 hostmice.

The mice in Group I (control) are thereafter given injections of 0.1 mlsterile saline every third day. The mice in Group II are thereaftergiven intradermal injections of 2 fg of synthetic YG of Example 20 in0.1 ml of sterile saline, every third day. The mice weigh approximately20 g each, so that 2 fg represents approximately 100 fg/kg ofbodyweight.

All of the mice are dead after approximately three weeks. The meansurvival rate of the Group II mice is approximately 2.4 days longer thanthat of the Group I mice.

Synthetic YG has an immunological activity similar to that of endogenousYG-material. However, the synthetic compound appears to be less potentthan the endogenous material. If the human-derived product wereidentical to the synthetic commercial chemical, this difference inactivity would not occur. Since the difference did occur, that factindicates that there must be some difference between the synthetic andendogenous products.

The discrepancy may be due to any of several possible causes. Thesynthetic commercial product may in whole or part be different isomericforms from the endogenous products. The endogenous materials may becomplexed, as discussed above, for example, with trace metal ions, suchas Zn++. The endogenous materials may be more stable than the chemicalmaterials, in vivo, because of chemical differences, or because of thepresence of other endogenous materials. While the synthetic commercialproduct is simply the dipeptide, per se, rather than derivativesthereof, the endogenous products may contain substitutions such as amethyl, acetyl, or amide for an H, or other molecular variations mayoccur, which are not capable of being detected by the analyticprocedures utilized here. Other endogenous materials accompanying theintrinsically active material may include phenylalanine, Phe--Ser,Gly--Gly, and Gly--Glu. (These have been found present in IMREG-1.)

It should be noted, however, that while the synthetic products purifiedfrom commercially available chemicals appear to be less activebiologically than the endogenous products, they are considerably cheaperto prepare. It is probably that the difference in activity is not adecisive factor in itself from a therapeutic standpoint; the syntheticproducts are believed more uniform in activity, from preparation topreparation, thereby permitting more reproducible (repeatable) results.The synthetic products are also free of the stigma attached toblood-derived products, which are often (correctly or incorrectly)believed by many persons to harbor viruses and other pathogens. Thelatter factors favoring the synthetic products may well be moreimportant, on balance, from a medical standpoint, thus offsetting anyrelatively lower biological activity.

Consequently, there may be a tradeoff of factors leading to a conclusionthat the chemically-derived synthetic products are biologicallyadequate, and because of their other advantages are therefore the basisof a preferred commercial embodiment. However, at this time it is notpossible to ascertain definitely whether the endogenous or syntheticembodiment is preferable.

B. Preparation and Assays of Synthetic Materials Chemically Related toYG and Other YG-material

It is believed that further synthetic versions of the endogenousYG-material described herein will be developed on the basis of furtheranalytic work built on the information disclosed herein. It is furtherbelieved that such synthetic materials, when purified from unacceptablecontaminants, may be considered preferable to the natural materialsderived from human leukocyte extracts. For example, it is known thatabsorption rate and blood level of pharmaceuticals can be improved bymolecular modifications of a natural or earlier discovered form of aproduct. The historical literature contains numerous instances. Thus,chlortetracycline was deschlorinated to provide an improved tetracyclineproduct; a bond in clorothiazide was saturated to producehydrochlorothiazide, thereby lowering the effective dosage amount by afactor of ten; and numerous synthetic forms of penicillin were providedby modifying the side chain attached to the beta lactam ring, such as byplacing a benzyl at its end of replacing an H attached to the side chainwith a CH₃ or NH₂, thereby making the product harder to hydrolyze (byaction of pencillinase) and otherwise providing desired characteristicsnot present in the original molecule.

In this connection, the paper of Schwartz et al., op. cit. supra(Background section of specification), summarizing various papers oninhibition of enzymatic cleavage of polypeptides, should be considered.Schwartz summarizes various papers that teach the addition ofappropriate moieties to inhibit hydrolysis and inactivation of productscontaining the peptide sequence H₂ N--Tyr--Gly--Gly-- . . . --COOH.These papers indicate that N-methylating the Tyr residue inhibitsaminopeptidase hydrolysis and cleavage of the Tyr--Gly amide bond. Theyalso indicate similar use of amidification, esterification, andreplacement with an alcohol of the C-terminal carboxyl. In addition,they indicate similar use of placing a D-aminoacid residue at or towardthe C-terminal end or inserting such a group (such as D-Ala or D-Met)between the Tyr and Gly residues. The inventor does not claim to havediscovered these expedients in the context of enkephalins, where theyare part of the prior art.

The inventor points out, however, that until the inventor's presentdiscoveries there has been no motivation for anyone to apply to thedipeptide YG these teachings concerning enkephalins. The reason is thatthere was no benefit to be gained thereby. In fact, the prior art (whichis exemplified by Schwartz, op. cit. supra) has taught that Yg is a"biologically inactive" and useless fragment or metabolite ofenkephalin; hence, nobody would have a reason to want to preventenzymatic cleavage of YG in a pharmaceutical product. Only after thepresent inventor discovered the immunological utility of YG did thereexist any motivation to prevent enzymatic cleavage of YG in apharmaceutical product.

EXAMPLE 25 Purification of Tyr-D-Ala-Gly

Commercial grade Tyr-D-Ala-Gly (L-tyrosyl-D-alanyl-L-glycine, SigmaChem. Co., St. Louis, Mo.) was purified in accordance with the procedureof Example 19. The elutant, at approximately 19.00 to 19.20 min, waspure Tyr--D--Ala--Gly, substantially free of contaminants (such aspyrogens and endotoxin).

EXAMPLE 26 DH Assay of Tyr-D-Ala-Gly

The DH assay of Example 21 is repeated with 1:10 serial dilutions ofTyr--D--Ala--Gly of Example 25, beginning with 300 fM. The amplifiereffect is slightly better than that observed in Table 1 of Gottlieb U.S.Pat. No. 4,699,898.

As an additional control, 0.1 ml of 3 fM Tyr--D--Ala--Gly is injectedwithout TT. No reaction is observed over 48 hours.

EXAMPLE 27 Gamma Interferon Assay of Tyr--D--Ala--Gly

The gamma interferon assay of Example 10 was repeated withTyr--D--Ala--Gly of Example 25.

For 1.0 L_(f) /ml TT, the maximum production was found to beapproximately 1.5 to 3 times the baseline amount. The Tyr--D--Ala--Glyconcentration was approximately 20 fM at the maximum production level.

The result observed in Example 27 was a production multiplier effectapproximately equivalent to that of endogenous YG-material, but itoccurred at a reagent concentration approximately an order of magnitudelower than for endogenous YG-material. That is, Tyr--D--Ala--Gly appearsto have been an order of magnitude more potent than endogenousYG-material in this assay. However, the human sources of the materialsused were not identical in the two examples. Thus, the observed datasuggests, but does not prove, that insertion of D--Ala into synthetic YGincreases the potency of the product over that of the endogenous YGProduct. (Also, the previously described greater potency of theendogenous YG Product over the synthetic YG Product should be noted.).The mechanism is attributed to inhibition of enzymatic degradation ofthe active molecule (i.e., increased resistance to hydrolysis of themolecule).

EXAMPLE 27A T-helper Cell IL-2 Receptor Expression Assay ofTyr-D-Ala-Gly

The T-helper cell receptor expression assay of Example 9 was repeatedwith Tyr--D--Ala--Gly of Example 25.

The highest figure for high-density of receptors was at approximately 2fM, and reflected an increase of IL-2 receptors by a factor ofapproximately 1.5 as a result of Tyr--D--Ala--Gly.

The result in Example 27A was approximately in order of magnitude lessin effect than endogenous YGG-material, but it occurred at an apparentlylower reagent concentration. Again, however, the two examples useddifferent human-source materials. This preliminary data suggests that aslightly greater range of dosage amounts is appropriate for YG expandedby insertion of a D-aminoacid. The inventor therefore considers that thedosage range of 10⁻²⁰ moles/kg to 1 fM/kg is appropriate for expandedYG-material.

EXAMPLE 28 Mouse Tests of Tyr--D--Ala--Gly

Ten BDF₁ strain mice are divided into two groups of 5. Each mouse isinjected with 100 L1210 leukemia cells from ascitic fluid of DBA/2 hostmice.

The mice in Group I (control) are thereafter given injections of 0.01 mlsterile saline every third day. The mice in Group II are thereaftergiven intradermal injections of 2 fg of synthetic Tyr--D--Ala--Gly ofExample 25 in 0.01 ml of sterile saline, every third day. The mice weighapproximately 20 g each, so that 2 fg represents approximately 100 fg/kgof bodyweight.

All of the mice are dead after approximately three weeks. The meanssurvival rate of the Group II mice is approximately 2.8 days longer thanthat of the Group I mice.

EXAMPLE 29 DH Assay of Tyr--D--Met--Gly

Tyr--D--Met--Gly is prepared by the method of Coy U.S. Pat. No.4,127,534, Example 20. It is purified by the procedure of above Example20, and the DH assay of above Example 26 is carried out, substitutingthe same molar amounts of the instant product.

Similar results are observed.

EXAMPLE 30 DH Assay of Tyr--D--Thr--Gly

Tyr--D--Thr--Gly is prepared by the method of Coy U.S. Pat. No.4,127,534, Example 18. It is purified by the procedure of above Example20, and the DH assay of above Example 26 is carried out, substitutingthe same molar amounts of the instant product.

Similar results are observed.

EXAMPLE 31 DH Assay of Tyr-D-Leu-Gly

Tyr--D--Leu--Gly is prepared by the method of Coy U.S. Pat. No.4,127,534, Example 4. It is purified by the procedure of above Example20, and the DH assay of above Example 26 is carried out, substitutingthe same molar amounts of the instant product.

Similar results are observed.

EXAMPLE 32 DH Assay of Try-D-Ile-Gly

Tyr--D--Ile--Gly is prepared by the method of Coy U.S. Pat. No.4,127,534, Example 6. It is purified by the procedure of above Example20, and the DH assay of above Example 26 is carried out, substitutingthe same molar amounts of the instant product.

Similar results are observed.

EXAMPLE 33 DH Assay of YG Hydrochloride

Commercial grade YG is reacted with HCl to produce YG hydrochloride. Theproduct is purified by the procedure of Example 20, and the DH assay ofExample 21 is carried out, substituting the same molar amounts of theinstant product as that of YG in Example 21.

Similar results are observed.

EXAMPLE 34 DH Assay of YG Citrate

Commercial grade YG is reacted with citric acid to produce YG citrate.The product is purified by the procedure of Example 20, and the DH assayof Example 21 is carried out, substituting the same molar amounts of theinstant product as that of YG in Example 21.

Similar results are observed.

EXAMPLE 35 DH Assay of YG Nitrate

Commercial grade YG is reacted with nitric acid to produce YG nitrate.The product is purified by the procedure of Example 20, and the DH assayof Example 21 is carried out, substituting the same molar amounts of theinstant product as that of YG in Example 21.

Similar results are observed.

EXAMPLE 36 DH Assay of YG Acetate

Commercial grade YG is reacted with acetic acid to produce YG acetate.The product is purified by the procedure of Example 20, and the DH assayof Example 21 is carried out, substituting the same molar amounts of theinstant product as that of YG in Example 21.

Similar results are observed.

EXAMPLE 37 DH Assay of Acetylated YG

Commercial grade YG is reacted with acetic anhydride to acetylate theYG, thereby producing n-acetyl tyrosylglycine. The product is purifiedby the procedure of Example 20, and the DH assay of Example 21 iscarried out, substituting the same molar amounts of the instant productas that of YG in Example 21.

Similar results are observed.

EXAMPLE 38 DH Assay of YG Inhibited with Bacitracin

10 ug of synthetic YG of Example 20 are dissolved in 50 ul of sterilesaline. 100 ug of bacitracin (Aldrich Chem. Co., Milwaukee, Wis., 50,000units/g) are added and thoroughly mixed.

The DH assay of Example 21 is carried out, using dilutions of theinstant mixture containing the same molar amounts of YG as in Example21.

Similar results are observed. There may be a prolongation of induration.

EXAMPLE 39 DH Assay of YG Inhibited with Puromycin

10 ug of synthetic YG of Example 20 are dissolved in 50 ul of sterilesaline. 100 ug of puromycin dichloride hydrate (Aldrich Chem. Co.,Milwaukee, Wis.) are added and thoroughly mixed.

The DH assay of Example 21 is carried out, using dilutions of theinstant mixture containing the same molar amounts of YG as in Example21.

Similar results are observed. There may be a prolongation of induration.

EXAMPLE 40 DH Assay of YG Inhibited with Amastatin

10 ug of synthetic YG of Example 20 are dissolved in 50 ul of sterilesaline. 100 ug of synthetic amastatin hydrochloride (Sigma Chem. Co.,St. Louis, Mo.) are added and thoroughly mixed.

The DH assay of Example 21 is carried out, using dilutions of theinstant mixture containing the same molar amounts of YG as in Example21.

Similar results are observed. There may be a prolongation of induration.

EXAMPLE 41 DH Assay of N-Methylated YG

Commercial grade YG is reacted with HCHO at pH 6.5 in phosphate bufferreduced with NaCNBH₄ to produce N-methylated YG. The product is purifiedby the procedure of Example 20, and the DH assay of Example 21 iscarried out, substituting the same molar amounts of the instant productas that of YG in Example 21.

Similar results are observed. There may be a prolongation of induration.

EXAMPLE 42 DH Assay of C-amidified YG

Commercial grade YG is linked by means of a benzyl ester to a Merrifieldresin. This is cleaved with NH₃ to yield C-amidified YG. The product ispurified by the procedure of Example 20, and the DH assay of Example 21is carried out, substituting the same molar amounts of the instantproduct as that of YG in Example 21.

Similar results are observed. There may be a prolongation of induration.

EXAMPLE 43 DH Assay of C-esterified YG

Commercial grade YG is reacted with Methanolic-HCl at 56° for 10 minutesto ethyl esterify the C-terminal carboxyl group of the Gly residue. Theproduct is purified by the procedure of Example 20, and the DH assay ofExample 21 is carried out, substituting the same molar amounts of theinstant product as that of YG in Example 21.

Similar results are observed. There may be a prolongation of induration.

EXAMPLE 44 DH Assay of C-alcoholated YG

Commercial grade YG is bound to a Merrifield resin, and reductivelycleaved with lithium borohydride in tetrahydrofuran. This processreplaces the C-terminal carboxyl group of the Gly residue with ethylalcohol. The product is purified by the procedure of Example 20, and theDH assay of Example 21 is carried out, substituting the same molaramounts of the instant product as that of YG in Example 21.

Similar results are observed. There may be a prolongation of induration.

An alternative method of delivering YG into the bloodstream or at a sitewhere it acts as a lymphokine is to administer a related molecule thatmetabolizes to YG in the body. Schwartz et al., op. cit. supra(Background section of this specification), describe how a polypeptideis enzymatically cleaved to moieties, such as Tyr--Gly, that are allegedto be "biologically inactive" or otherwise useless (which theyapparently are, for the purposes of Schwartz et al. and those whose workthey describe). They describe how injection of Leu- and Met-enkephalininto rats and mice has resulted int he production of, among otherthings, Tyr--Gly metabolites. They indicate that purifiedangiotensin-converting enzyme (hereinafter referred to as ACE) anddipeptidylcarboxypeptidase (hereinafter referred to as DPCP) cleave aGly--Phe amide bond in these polypeptides, producing a Tyr--Gly--Glymoiety; and that the Gly--Gly amide bond is cleaved bydipeptidylaminopeptidase, producing a Tyr--Gly moiety. They alsoidentify ACE as a form of DPCP, and finally conclude that an enzymedesignated as "enkephalin-DPCP" or "enkephalinase" is the principalenzyme that metabolizes the polypeptides of interest to them into"biologically inactive" moieties such as Tyr--Gly and Tyr--Gly--Gly. The"biologically inactive" moieties of no interest to Schwartz et al. areproducts of interest in connection with the instant invention.

It is now within the state of the art to synthesize desired polypeptidesby means of an automatic peptide synthesizer, such as the Beckman Model990. Moreover, as noted in the Background section of the specification,it is known to deliver a therapeutically active molecule byadministering another, larger molecule that hydrolyzes, by reason ofaction of endogenous enzymes, within the body to the desired molecule.

These facts, when considered with the novel information first disclosedin this specification, indicate that a way to deliver YG within the bodyis to administer Tyr--Gly--A or Tyr--Gly--Gly--B, where A and Brepresented one or more additional amino acid residues, such as Phe,Phe--Met, Phe--Leu, or a longer amino acid residue sequence. The productso administered must be one susceptible to hydrolysis by one or moreendogenous enzymes, for example, as enkephalins are so hydrolyzed.Preferably, the product so administered is not an endogenous enkephalinor endorphin, or other opiate-related product. That is so that possiblepsychotropic or other physiological effects of such products will beavoided, as well as regulatory and legal problems associated with usingdesigner drugs potentially subject to abuse because of their opiodeffects.

This method of delivering YG is not at this time considered a preferredembodiment so much as an expedient that a would-be in-fringer wouldattempt to avoid the scope of any claims directed solely to Tyr--Gly.Such an expedient might avoid literal infringement, while at the sametime appropriating the gist of the invention and the inventor'sdiscoveries nd teachings set forth hereinabove. Such expedients aretherefore considered within the scope of the instant invention as beingin the general nature of equivalents, albeit less preferred and lesseffective embodiments.

However, the following caution must be observed in connection with thistype of expedient: There is as yet no scientifically valid basis forconcluding that a metabolite of a polypeptide is molecularly identicalto a smaller peptide that has the same amino acid residues, but isproduced by means of a different procedure. For example, consider themetabolites of Met-enkephalin described by Schwartz et al. op. cit.supra. Such a metabolite may be identified as having a Tyr-Gly aminoacid residue sequence. Yet, there is no clear basis for equating themolecular structure of the metabolite with Yg-material endogenouslymanufactured within the human body. The YG metabolite and the endogenousYG-material may differ in an indefinite number of possible ways: forexample, one or the other may be amidified, methylated, acetylated,esterified; different isomeric forms or dimers may be involved; metalliccomplexes may exist; one may have side chains that the other does not.Whether such difference exist and whether they are clinically importantwould have to be ascertained, and the necessary technology for doingthat may be beyond the present state of the art. Hence, it must beregarded as not scientifically established (and perhaps not possible toestablish at this time, given the state of the art) whether a Tyr--Glymetabolite of a polypeptide such as Tyr--Gly--Gly--Phe-- . . . (or ofany other Tyr--Gly-- . . . polypeptide) is molecularly identical toendogenous YG-material (Beta-1.11).

EXAMPLE 45 DH Assay of Nonendogenous Polypeptide Susceptible toEnzymatic Hydrolysis

Tyr--Gly--Gly--Phe--Ile is prepared in a Beckman Model 990 automaticpeptide synthesizer using conventional techniques. The product ispurified by the procedure of Example 20.

The DH assay of Example 21 is carried out, substituting for the YGdilutions of the Example the same molar concentrations of the instantnonendogenous polypeptide. Significant immunological activity is notobserved.

The procedure is repeated using serial 10:1 greater concentrations.Activity comparable to that of YG appears to exist at approximately 8-10orders of magnitude greater molar concentration of polypeptide productthan YG in Example 21.

The activity may be attributed to enzymatic cleavage of the Gly--Glybond by dipeptidylaminopeptidase, producing a Tyr--Gly metabolite. (ACEor enkephalinase cleavage of the Gly--Phe bond to produce Tyr--Gly--Glyis another possibility.)

It is considered that an effective dosage amount in this context must beone that will yield approximately the same amount of YG-material in thebody as would constitute a correct dosage amount of the latter (i.e.,approximately from 7 to 70 fg of YG per kg).

GENERAL CONCLUDING REMARKS

The above described endogenous amplifiers of the immune system areconsidered to be materials whose natural function is regulation of theimmune response, directly with respect to cell-mediated immunity andperhaps indirectly affecting humoral immunity as well. The materialshave been prepared with a high degree of purity such that theirmolecular structure has been more fully characterized than hithertopossible. Furthermore, the materials of this invention have beenpurified sufficiently to permit their administration to human subjectsto produce beneficial effects, without known harmful side effects. Suchbeneficial effects include immunostimulation of immunodeficientpatients, such as victims of AIDS and ARC, and retardation of the normalprogression to AIDS from ARC. While not curative of AIDS and ARC, thematerials of the invention have substantial therapeutic effects,alleviating certain AIDS/ARC symptoms and reversing or slowing someharmful effects of the diseases. For example, candidiasis has been shownin the above data to be alleviated by this treatment. In addition,synthetic products have been described with molecular structures basedon that of the endogenous materials, and possessing similarimmunological utility.

The products described above have been shown to increase a person'sendogenous production of various modifiers of biological responses thathuman lymphocytes produce, at least when the person's immune system hasnot already been so injured as to be beyond the reach of therapy. Suchleukocyte-generated modifiers of biological response have been shown toinclude the specific lymphokines IL-2 and gamma-interferon.

While the invention has been described in connection with specific andpreferred embodiments thereof, it is capable of further modificationswithout departing from the spirit and scope of the invention. Thisapplication is intended to cover all variations, uses, or adaptations ofthe invention, following, in general, the principles of the inventionand including such departures from the present disclosure as come withinknown or customary practice within the art to which the inventionpertains.

As used in the claims "recall antigen" refers to an antigen to which aperson or other subject has previously been exposed.

As the preceding examples and discussion show, the invention can bepracticed with a genus of products characterized by the presence of Tyrand Gly amino acid residues, with optional admixture with other productsand with optional modification of certain parts of the structure. Asused in the claims, the following terms have the following meanings:

"YG Product" means YG or methylated, acetylated, amidified, esterified,or alcoholated YG; or a pharmaceutically acceptable salt of any of theforegoing products.

"Inhibited YG Product" means a YG Product mixed with bacitracin,puromycin, amastatin, or an equivalent means for inhibing enzymatichydrolysis of the Tyr--Gly bond.

"Expanded YG Product" means Tyr--X--Gly, where X is a D-amino-acid; orthe foregoing product methylated, acetylated, amidified, esterified, oralcoholated; or a pharmaceutically acceptable salt of any of theforegoing products.

"YGG Product" means YGG or methylated, acetylated, amidified,esterified, or alcoholated YGG; or a pharmaceutically acceptable salt ofany of the foregoing products.

The subject matter claimed is:
 1. A method for slowing clinicalprogression to AIDS in an ARC patient, comprising administering to saidpatient an effective dosage amount of YG Product, said YG Product beingYG; methylated, acetylated, amidified, esterified, or alcoholated YG; ora pharmaceutically acceptable salt of any of the foregoing.
 2. Themethod of claim 1 wherein said amount, administered biweekly bysubcutaneous or intradermal injection, is that which is derived fromapproximately 125,000 leukocytes.
 3. The method of claim 2 wherein YGGProduct is concurrently administered to said patient, said YGG Productbeing YGG; methylated, acetylated, amidified, esterified, or alcoholatedYGG; or a pharmaceutically acceptable salt of any of the foregoing. 4.The method of claim 3 wherein the ratio of the amount of said YG Productto the amount of YGG Product is more than 10:1 and less than 25:1 (w/w).5. The method of claim 1 wherein phenylalanine is concurrentlyadministered to said patient.
 6. The method of claim 5 wherein at leastone of the following is concurrently administered to said patient:Phe--Ser, Gly--Gly, Gly--Glu.
 7. A method of treating a person forcandidiasis, comprising administering to said person an effective dosageamount of said YG Product.
 8. The method of claim 7 wherein saidcandidiasis is oral candidiasis.
 9. A method for alleviating at leastone of the clinical symptoms of a person suffering from AIDS or ARC,said method comprising administering to said person an effective dosageamount of YG Product, and said symptoms comprising decrease inpercentage of T-helper cells, weight loss, diarrhea, tissue breakdowncharacterized by increase in serum level of uric acid or creatinephosphokinase, and candidiasis, said YG Product being YG; methylated,acetylated, amidified, esterified, or alcoholated YG; or apharmaceutically acceptable salt of any of the foregoing.
 10. The methodof claim 9 wherein said amount, administered biweekly by subcutaneous orintradermal injection, is from 3×10⁻¹⁸ moles/kg to 3 f moles/kg.
 11. Themethod of claim 9 wherein said amount of said YG Product, administeredbiweekly by subcutaneous or intradermal injection, is from 0.5 fg/kg to500 fg/kg.
 12. The method of claim 9 wherein an effective dosage amountof said YGG Product is also administered to said person, said YGGProduct being YGG; methylated, acetylated, amidified, esterified, oralcoholated YGG; or a pharmaceutically acceptable salt of any of theforegoing.
 13. The method of claim 12 wherein the dosage amount of saidYG Product and the dosage amount of said YGG Product are in the ratio ofapproximately 18:1 (w/w).
 14. The method of claim 9 wherein said YGProduct is administered biweekly by subcutaneous or intradermalinjection in a dosage amount of from 0.5 fg/kg to 500 fg/kg, and whereinYGG Product is also administered biweekly by subcutaneous or intradermalinjection in a dosage amount of from 0.01 fg/kg to 100 fg/kg.
 15. Amethod for increasing immune system response of a mammalian subject torecall antigen, comprising administering to said subject an effectivedosage amount of said YG Product, said YG Product being YG, methylated,acetylated, amidified, esterified, or alcoholated YG; or apharmaceutically acceptable salt of any of the foregoing.
 16. The methodof claim 15 where said subject is human.
 17. The method of claim 15where said subject is immunodeficient.
 18. A method for increasingimmune system response of a mammalian subject to recall antigen,comprising administering to said subject an effective dosage amount ofInhibited YG Product, said Inhibited YG Product being said YG Productmixed with bacitracin, puromycin, amastatin, or another means forinhibiting enzymatic hydrolysis of a Tyr--Gly bond.
 19. The method ofclaim 18 where said subject is human.
 20. The method of claim 18 wheresaid subject is immunodeficient.
 21. A method for increasing immunesystem response of a mammalian subject to recall antigen, comprisingadministering to said subject an effective dosage amount of Expanded YGProduct, said Expanded YG Product being Tyr--X--Gly where X is aD-aminoacid; or the foregoing product methylated, acetylated, amidified,esterified, or alcoholated; or a pharmaceutically acceptable salt of anyof the foregoing.
 22. The method of claim 21 where said subject ishuman.
 23. The method of claim 21 where said subject is immunodeficient.24. The method of claim 22 wherein said amount, administered biweekly bysubcutaneous or intradermal injection, is at least 3×10⁻¹⁹ moles/kg andis no more than 3 f moles/kg.
 25. The method of claim 21 wherein saidexpanded YG Product is Tyr--D--ala--Gly.
 26. A method for increasingimmune system response of a mammalian subject to recall antigen,comprising administering to said subject an amount of a nonendogenouspolypeptide that, as a result of action of at least one endogenousenzyme, metabolizes to an effective dosage amount of YG Product.
 27. Themethod of claim 26 wherein said dosage amount of YG Product is from3×10⁻¹⁸ moles/kg to 3 f moles/kg.
 28. A method for increasing a person'sendogenous production of modifiers of biological responses produced inleukocytes, comprising administering to said person an effective dosageamount of said YG Product.
 29. The method of claim 28 wherein saidmodifier is a lymphokine.
 30. The method of claim 29 wherein saidlymphokine is IL-2.
 31. The method of claim 29 wherein said lymphokineis gamma-interferon.
 32. A method for increasing immune system responseof a mammalian subject to recall antigen to which said subject haspreviously been exposed, comprising administering to said subject aneffective dosage amount of a purified human leukocyte dialysate,purified of substantially all endotoxin and pyrogen, comprisingendogenous material possessing intrinsic immunoamplifier activity, wheresaid endogenous material is YG-material or YGG-material, said YG Productbeing YG; methylated, acetylated, amidified, esterified, or alcoholatedYG; or a pharmaceutically acceptable salt of any of the foregoing; andsaid YGG Product being YGG; methylated, acetylated, amidified,esterified, or alcoholated YGG; or a pharmaceutically acceptable salt ofany of the foregoing.
 33. The method of claim 32 wherein said subject ishuman.
 34. The method of claim 32 wherein said subject isimmunodeficient.
 35. The method of claim 32 wherein said endogenousmaterial consists of endogenous said YG Product.
 36. The method of claim33 wherein said effective dosage amount is from 3×10⁻¹⁸ moles/kg to 3 fmoles/kg.